Fibronectin based scaffold domain proteins that bind to myostatin

ABSTRACT

The present invention relates to fibronectin-based scaffold domain proteins that bind to myostatin. The invention also relates to the use of these proteins in therapeutic applications to treat muscular dystrophy, cachexia, sarcopenia, osteoarthritis, osteoporosis, diabetes, obesity, COPD, chronic kidney disease, heart failure, myocardial infarction, and fibrosis. The invention further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the proteins.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/632,436, filed Feb. 26, 2015 (now U.S. Pat. No. 9,662,373), which isa divisional of U.S. patent application Ser. No. 14/275,542, filed May12, 2014 (now U.S. Pat. No. 8,993,265 B2), which is a continuation ofU.S. patent application Ser. No. 14/025,253, filed Sep. 12, 2013 (nowU.S. Pat. No. 8,933,199 B2), which claims priority to U.S. ProvisionalApplication No. 61/700,697 entitled “Fibronectin based scaffold domainproteins that bind to myostatin” filed on Sep. 13, 2012, and U.S.Provisional Application No. 61/780,005 entitled “Fibronectin basedscaffold domain proteins that bind to myostatin” filed Mar. 13, 2013.The contents of the aforementioned applications are hereby incorporatedby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 19, 2017, isnamed MXI_514ACNDVCN_Sequence_Listing.txt and is 251,219 bytes in size.

FIELD OF THE INVENTION

The present invention relates to fibronectin-based scaffold domainproteins that bind myostatin. The invention also relates to the use ofthe innovative proteins in therapeutic applications to treatmuscle-wasting diseases and metabolic disorders. The invention furtherrelates to cells comprising such proteins, polynucleotides encoding suchproteins or fragments thereof, and to vectors comprising thepolynucleotides encoding the innovative proteins.

BACKGROUND OF THE INVENTION

Myostatin, also known as growth and differentiation factor-8 (GDF-8), isa member of the transforming growth factor-β (TGF-β) superfamily ofsecreted growth factors. Myostatin has all of the structural featurescommon to the TGF-1 family proteins: a hydrophobic amino-terminus thatacts as a secretory signal, nine invariant cysteine residues, and an“RXXR” furin-type proteolytic processing site. Proteolytic cleavage ofthe protein gives rise to a C-terminal domain which forms a homodimerthat is the biologically active form of myostatin (Thies et al., GrowthFactors 2001; 18(4):251-9). Alignments of the C-terminal fragment ofmyostatin amino acid sequences from multiple vertebrate species revealthat the protein is highly conserved (100% identity) between human,monkey, cow, dog, mouse, rat, turkey and chicken (McPherron, et al.PNAS, 94:12457-61, 1997).

Myostatin expression is limited primarily to skeletal muscle and adiposetissue, where it has been shown to be a negative regulator of skeletalmuscle development (Lee L S, Immunol Endocr Metab Agents Med Chem. 2010;10:183-194). In mammals, skeletal muscle appears to be the principaltarget tissue of myostatin, where it binds to cell-surface receptors,leading to muscle loss. Mice and cattle with genetic deficiencies inmyostatin exhibit dramatic increases in skeletal muscle mass, i.e., the“double muscling” phenotype, therefore supporting the role of myostatinin suppressing muscle growth (McPherron and Lee, Proc Natl Acad Sci USA.2003 Dec. 23; 100(26):15842-6). Muscle hypertrophy in Belgian Blue andPiedmontese cattle breeds is due to a missense mutation within the thirdexon of the bovine myostatin gene (Bass et. al., Domest Anim Endocrinol.1999; 17(2-3):191-7). Transgenic overexpression of myostatin inhibitorsalso results in hyper-muscularity. Enhanced muscle growth in theseanimals is due to an increase in both cell number, or hyperplasticgrowth, and cell size, or hypertrophic growth, which results in largerand heavier myofibers. Increased skeletal muscle mass due to a myostatinmutation has also been reported in humans. Myostatin inhibitioneffectively increases skeletal muscle mass and strength, both in thepostnatal period and in adults.

Increases in skeletal muscle mass and strength are also associated withmetabolic adaptations which positively affect body composition, energyexpenditure, glucose homeostasis and insulin requirements. Both geneticand pharmacological findings indicate that myostatin regulates energymetabolism and that its inhibition can significantly attenuate theprogression of metabolic diseases, including obesity and diabetes. Forexample, myostatin null mice exhibit decreased body fat accumulation(McPherron & Lee, J. JCI 109:595, 2002) when compared with wild typemice of the same age. This reduction in body fat is a manifestation ofreduced adipocyte number and size, implicating a significant role ofmyostatin in adipogenesis as well as in myogenesis.

Accordingly, myostatin is a desirable target for therapeutic orprophylactic intervention for the treatment of disorders or conditionswhich would benefit from an increase in muscle mass, muscle strengthand/or metabolism (e.g., muscular dystrophy, frailty, disuse atrophy andcachexia), disorders associated with muscle wasting (e.g., renaldisease, cardiac failure or disease, and liver disease), and metabolicdisorders (e.g., Type II diabetes, metabolic syndrome, obesity andosteoarthritis).

Accordingly, it would be advantageous to obtain improved fibronectindomain scaffold proteins that bind myostatin for the therapeutictreatment of, e.g., metabolic disorders, muscle wasting disorders, andmuscle loss due to inactivity.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofAdnectins that bind to and antagonize myostatin. Specifically, theanti-myostatin Adnectins of the present invention inhibit myostatinactivity, thereby affecting downstream SMAD signaling. One mechanismaccounting for altered SMAD signaling of some of the anti-myostatinAdnectins of the invention involves the inhibition of Alk4 recruitmentto the myostatin-ActRIIb complex, the physiological consequences ofwhich are increased muscle volume and body weight.

In one aspect, the invention provides a polypeptide comprising afibronectin tenth type III domain (¹⁰Fn3) wherein the ¹⁰Fn3 has at leastone loop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain, and wherein the polypeptide binds myostatin. In certainembodiments, the polypeptide binds myostatin with a K_(D) of less than500 nM.

In some embodiments, the BC loop of the polypeptide of the inventioncomprises an amino acid sequence according to the formulaX₁-L-P-X₂-X₃-X₄-X₅-X₆-X₇, wherein (a) X₁ is selected from the groupconsisting of S, T and Y; (b) X₂ is selected from the group consistingof H, Y, N, R, F, G, S and T; (c) X₃ is selected from the groupconsisting of A, P, Q, S, F, H, N and R; (d) X₄ is selected from thegroup consisting of G and A; (e) X₅ is selected from the groupconsisting of H, L, R, V, N, D, F, I and K; (f) X₆ is selected from thegroup consisting of A, L, G, M, F, I and V; and (g) X₇ is selected fromthe group consisting of H and N. In certain embodiments, X₁ is S, and/orX₂ is H or Y, and/or X₃ is A or P, and/or X₄ is G, and/or X₅ is H, L orR, and/or X₆ is A or L, and/or X₇ is H. In other embodiments, the BCloop comprises an amino acid sequence according to the formulaX₁₉-X₂₀-P-X₂₁-G-X₂₂-A, wherein (a) X₁₉ is selected from the groupconsisting of D, E, V and W; (b) X₂₀ is selected from the groupconsisting of A, S and V; (c) X₂₁ is selected from the group consistingof R, A, G, K and L; and (d) X₂₂ is selected from the group consistingof L and R. In certain embodiments, X₁₉ is D, and/or X₂₀ is A, S or V,and/or X₂₂ is L.

In some embodiments, the DE loop comprises an amino acid sequenceaccording to the formula G-R-G-X₈, wherein X₈ is V or L.

In some embodiments, the DE loop comprises an amino acid sequenceaccording to the formula X₂₃-G-R-G-X₂₄, wherein (a) X₂₃ is selected fromthe group consisting of V, P, F, I and L; and (b) X₂₄ is selected fromthe group consisting of S, N and T.

In some embodiments, the FG loop of the polypeptide of the inventioncomprises an amino acid sequence according to the formulaX₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈, wherein (a) X₉ is selected fromthe group consisting of L, V and I; (b) X₁₀ is selected from the groupconsisting of T and S; (c) X₁₁ is selected from the group consisting ofK, R, A, G, S, D, H, N, T and P; (d) X₁₂ is selected from the groupconsisting of S, T, A, E, H, K and N; (e) X₁₃ is selected from the groupconsisting of K, G, Q, D, E, N, T and S; (f) X₁₄ is selected from thegroup consisting of V, I, F, L, M, P, T and Y; (g) X₁₅ is selected fromthe group consisting of I, L and Y; (h) X₁₆ is selected from the groupconsisting of H, I, V, K, L, R, F, G, S and T; (i) X₁₇ is selected fromthe group consisting of Y and H; and (j) X₁₈ is selected from the groupconsisting of K, M, L, R and V.

In certain embodiments, X₉ is L or V, and/or X₁₀ is T, X₁₁ is K or R,and/or X₁₂ is S or T, and/or X₁₃ is K, G or Q, and/or X₁₄ is V or I,and/or X₁₅ is I, and/or X₁₆ is H, I or V, and/or X₁₇ is Y and/or X₁₈ isK or M.

In other embodiments, the FG loop comprises an amino acid sequenceaccording to the formula X₂₅-X₂₆-R-X₂₇-G-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂, wherein(a) X₂₅ is selected from the group consisting of I and V; (b) X₂₆ isselected from the group consisting of F, D and Y; (c) X₂₇ is selectedfrom the group consisting of D and T; (d) X₂₈ is selected from the groupconsisting of P, M, V and T; (e) X₂₉ is selected from the groupconsisting of V, L, N, R and S; (f) X₃₀ is selected from the groupconsisting of H, T, L, N, Q and S; (g) X₃₁ is selected from the groupconsisting of F, W, Y, H and L; and (h) X₃₂ is selected from the groupconsisting of D, A and G.

In certain embodiments, X₂₅ is I, and/or X₂₆ is F, and/or X₂₇ is D,and/or X₂₈ is P, and/or X₂₉ is V, and/or X₃₀ is H or T, and/or X₃₁ is For W, and/or X₃₂ is D.

In some embodiments, the polypeptide comprises a BC loop and a DE loop,or a BC loop and FG loop, or a DE loop and an FG loop, or a BC loop, aDE loop and an FG loop. In some embodiments, the BC loop of thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7-38. In other embodiments, the DE loopcomprises an amino acid selected from the group consisting of SEQ IDNOs: 39-45. In yet other embodiments, the FG loop comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 46-79.In some embodiments, the BC, DE, or FG loop amino acid sequence is atleast 80% identical to any one of SEQ ID NOs: 7-38, 39-45, and 46-79,respectively. In other embodiments, the polypeptide comprises an aminoacid sequence that is at least 80% identical to any one of SEQ ID NOs:80-123, 228-239, and 252-273. In yet other embodiments, the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 80-123, 228-239, and 252-273.

In some embodiments, the polypeptides comprise the BC, DE, and FG loopcombinations as shown in Table 1. In one embodiment, the polypeptide hasthe BC, DE, and FG loops as set forth in SEQ ID NOs: 34, 39, and 75,respectively.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservativeamino acid substitutions. In some embodiments, the BC loop has an aminoacid sequence according to the formula X₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉,wherein X₃₃ is T or Y; X₃₄ is Y, N, R, F, G, S, or T; X₃₅ is A, P, S, F,H, N, or R; X₃₆ is A; X₃₇ is H, L, R, V, N, D, F, or I; X₃₈ is L, G, M,F, I, or V; and X₃₉ is H.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the DE loophas 1 amino acid substitution, such as a conservative amino acidsubstitution. In some embodiments, the DE loop has an amino acidsequence according to the formula G-R-G-X₄₀, wherein X₄₀ is L.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the FG loophas 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such asconservative amino acid substitutions. In some embodiments, the FG loophas an amino acid sequence according to the formulaX₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I; X₄₂ isS; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, or N; X₄₅is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ is I or Y;X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L, R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H, and the DEloop has an amino acid sequence according to the formula G-R-G-X₄₀,wherein X₄₀ is L.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H, and the FGloop has an amino acid sequence according to the formulaX₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I; X₄₂ isS; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, or N; X₄₅is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ is I or Y;X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L, R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the DE loophas an amino acid sequence according to the formula G-R-G-X₄₀, whereinX₄₀ is L, and the FG loop has an amino acid sequence according to theformula X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I;X₄₂ is S; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, orN; X₄₅ is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ isI or Y; X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L,R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H; the DE loophas an amino acid sequence according to the formula G-R-G-X₄₀, whereinX₄₀ is L; and the FG loop has an amino acid sequence according to theformula X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I;X₄₂ is S; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, orN; X₄₅ is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ isI or Y; X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L,R, or V.

In another embodiment, the polypeptide comprises an amino acid sequenceat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence set forth in SEQ ID NO: 273 [PRD-1474], SEQ ID NO:118 [3116_A06], or SEQ ID NO: 281 [core Adnectin sequence of PRD-1474and 3116_A06 preceded by N-terminal extension sequence (GVSDVPRDL) andfollowed by a C-terminal tail (EI)]. In yet another embodiment, thepolypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regionsof SEQ ID NO: 273, 118, or 281.

In some embodiments, the polypeptides described above may comprise oneor more pharmacokinetic (PK) moieties such as polyethylene glycol,sialic acid, Fc, Fc fragment, transferrin, serum albumin, a serumalbumin binding protein, and a serum immunoglobulin binding protein. Inone embodiment, the PK moiety is a serum albumin binding protein thatcomprises an ¹⁰Fn3 domain which binds to, for example, HSA. In anotherembodiment, the PK moiety is Fc and can be on either the N- orC-terminus of the polypeptide, and optionally form a dimer. In yet otherembodiments, the PK moiety is polyethylene glycol. In some embodiments,the PK moiety and polypeptide are linked via at least one disulfidebond, a peptide bond, a polypeptide, a polymeric sugar or a polyethyleneglycol moiety.

In another aspect, the invention provides a pharmaceutical compositioncomprising a polypeptide described above, which is optionallyendotoxin-free. In another aspect, the invention provides an isolatednucleic acid molecule encoding a polypeptide described above, anexpression vector comprising a nucleotide sequence, and a cellcomprising the nucleic acid encoding the polypeptide. In another aspect,the invention provides a method of producing the anti-myostatinpolypeptide by culturing the cell.

In another aspect, the invention provides a method of attenuating orinhibiting a myostatin-related disease or disorder in a subject byadministering an effective amount of the polypeptide or compositioncomprising a polypeptide described above. In some embodiments, thedisease to be treated is muscular dystrophy, amyotrophic lateralsclerosis, congestive obstructive pulmonary disease, chronic heartfailure, cancer, AIDs, renal failure, chronic kidney disease, uremia,rheumatoid arthritis, sarcopenia, muscle wasting due to prolongedbedrest, spinal cord injury, stroke, bone fracture, aging, diabetes,obesity, hyperglycemia, cachexia, osteoarthritis, osteoporosis,myocardial infarction, or fibrosis.

In another aspect, the invention provides a method of attenuating orinhibiting a disorder associated with degeneration or wasting of musclein a subject.

In another aspect, the invention provides a method of administering thepolypeptide to increase muscle mass, increase the number of musclecells, increase the size of muscle cells and/or increase musclestrength.

In another aspect, the invention provides a method of attenuating orinhibiting a metabolic disorder in a subject. In some embodiments, themetabolic disorder is diabetes (e.g., type II diabetes), hyperglycemia,hyperinsulinaemia, hyperlipidaemia, insulin resistance, impaired glucosemetabolism, obesity, or metabolic syndrome. In some embodiments, asecond therapeutic composition can be administered. In otherembodiments, administration of the polypeptide results in increasedinsulin sensitivity, increased glucose uptake by cells, decreased bloodglucose levels, and/or decreased body fat.

In another aspect, the invention provides a method for enhancing leanmuscle mass in a subject comprising administering an effective amount ofa polypeptide or composition described above.

In another aspect, the invention provides a method for increasing theratio of lean muscle mass to fat in a subject comprising administeringan effective amount of a polypeptide or composition described above.

In another aspect, the invention provides kits comprising a polypeptideor composition described above, and instructions for use.

In another aspect, the invention provides methods of detecting ormeasuring myostatin in a sample comprising contacting the sample with apolypeptide described above, and detecting or measuring binding of thepolypeptide to myostatin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of exemplary anti-myostatin Adnectin aminoacid sequences. The BC, DE, and FG loop amino acid sequences areidentified by underlining, italics/underlining or bold/underlining,respectively.

FIG. 2 depicts a WebLogo-based analysis of the varied residues of the BCloop of the 1979_B06 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the BC loop that werevaried during PROfusion. The image was created using WebLogo (Crooks GE, Hon G, Chandonia J M, Brenner S E. WebLogo: A sequence logogenerator. Genome Research 2004; 14:1188-1190).

FIG. 3 depicts a WebLogo-based analysis of the varied residues of the DEloop of the 1979_B06 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the DE loop that werevaried during PROfusion.

FIG. 4 depicts a WebLogo-based analysis of the varied residues of the FGloop of the 1979_B06 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the FG loop that werevaried during PROfusion.

FIG. 5 depicts a WebLogo-based analysis of the varied residues of the BCloop of the 2062_G02 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the BC loop that werevaried during PROfusion.

FIG. 6 depicts a WebLogo-based analysis of the varied residues of the DEloop of the 2062_G02 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the DE loop that werevaried during PROfusion.

FIG. 7 depicts a WebLogo-based analysis of the varied residues of the FGloop of the 2062_G02 family of anti-myostatin Adnectins. Indicated isthe frequency of amino acids in each position of the FG loop that werevaried during PROfusion.

FIG. 8 depicts a graph of a competitive binding assay (competitiveELISA) showing that Adnectins PRD-1285, PRD-1286, and PRD-1288 do notblock myostatin binding to ActRIIb-Fc. Indicated is the % competition ofActRIIb-Fc binding to myostatin. As expected, the positive controlActRIIb-Fc construct competed with ActRIIb-Fc binding to myostatin.

FIG. 9 depicts a graph showing the effects of various concentrations ofAdnectins PRD-1285, PRD-1286, and PRD-1288 on myostatin activity in anARE-luciferase assay. Experimental conditions are as described inExample 3. Each of PRD-1285, PRD-1286, and PRD-1288 inhibited 100% ofthe myostatin-induced ARE-luciferase activity.

FIG. 10 is a schematic depicting the mechanism of action by which theAdnectins of the present invention inhibit myostatin activity. Thenative signaling complex is shown in the left panel. Specifically,binding of myostatin to ActRIIb is followed by recruitment of activinreceptor-like kinase 4 (ALK4) or ALK5, and ActRIIb and ALK4/5 bind todistinct regions of myostatin. The Adnectins of the present inventionprevent the binding of ALK4/5, but not ActRIIb, to myostatin (rightpanel).

FIG. 11 depicts a bar graph showing percent increases in body weight atday 15 in mice treated with the indicated anti-myostatin Adnectinscompared to control mice. B6 SCID mice were treated either biweekly orweekly with subcutaneous injections of anti-myostatin Adnectins for 14days, as described in Example 10. Body weights were measured throughoutthe treatment period; calculated percentage change values for day 15 aredepicted. (*=indicates statistical differences from respective controlgroup; p≤0.01 t-test).

FIG. 12 depicts a bar graph showing increases in leg muscle volume (incm³) at day 15 in mice treated with the indicated anti-myostatinAdnectins compared to control mice. B6 SCID mice were treated eitherbiweekly or weekly with subcutaneous injections of anti-myostatinAdnectins for 14 days as described in Example 10. (*=indicatesstatistical differences from respective control group; p≤0.05 t-test).

FIG. 13 depicts a bar graph showing increases in leg muscle volume (incm³) at day 28 in mice treated with the various indicated doses ofPRD-1474. B6 SCID mice were treated either biweekly or weekly withsubcutaneous injections of PRD-1474 for 28 days as described in Example11. (*p<0.0001; # not significant between groups).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled artisan.Although any methods and compositions similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention, the preferred methods and compositions are described herein.

“Full-length myostatin” as used herein refers to the full lengthpolypeptide sequence described in McPherron et al. (1997), supra, aswell as related full-length polypeptides including allelic variants andinterspecies homologs. The term “myostatin” or “mature myostatin” refersto fragments of the biologically active mature myostatin, as well asrelated polypeptides including allelic variants, splice variants, andfusion peptides and polypeptides. The mature C-terminal protein has beenreported to have 100% sequence identity among many species includinghuman, mouse, chicken, porcine, turkey, and rat (Lee et al., PNAS 2001;98:9306). The sequence for human prepromyostatin is:

(SEQ ID NO: 1) MQKLQLCVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS

The sequence for human pro-myostatin is:

(SEQ ID NO: 2) NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINTMLYFNGKEQIIYGKIPAMVVDRC GCS

The sequence for mature myostatin (conserved in human, murine, rat,chicken, turkey, dog, horse, and pig) is:

(SEQ ID NO: 3) DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPA MVVDRCGCS.

“Polypeptide” as used herein refers to any sequence of two or more aminoacids, regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D). Thepeptides of the invention are proteins derived from the tenth type IIIdomain of fibronectin that have been modified to bind specifically tomyostatin and are referred to herein as, “anti-myostatin Adnectin” or“myostatin Adnectin.”

A “polypeptide chain”, as used herein, refers to a polypeptide whereineach of the domains thereof is joined to other domain(s) by peptidebond(s), as opposed to non-covalent interactions or disulfide bonds.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes the polypeptide in situ within recombinantcells since at least one component of the polypeptide's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR™) software. Those skilledin the art can readily determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared. For example, the %amino acid sequence identity of a given amino acid sequence A to, with,or against a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y where Xis the number of amino acid residues scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of A andB, and where Y is the total number of amino acid residues in B. It willbe appreciated that where the length of amino acid sequence A is notequal to the length of amino acid sequence B, the % amino acid sequenceidentity of A to B will not equal the % amino acid sequence identity ofB to A.

As used herein, the term “Adnectin binding site” refers to the site orportion of a protein (e.g., myostatin) that interacts or binds to aparticular Adnectin. Adnectin binding sites can be formed fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Adnectin binding sites formed bycontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas Adnectin binding sites formed by tertiary folding aretypically lost on treatment of denaturing solvents.

An Adnectin binding site for an anti-myostatin Adnectin of the inventionmay be determined by application of standard techniques typically usedfor epitope mapping of antibodies including, but not limited to proteasemapping and mutational analysis. Alternatively, an Adnectin binding sitecan be determined by competition assay using a reference Adnectin orantibody which binds to the same polypeptide, e.g., myostatin (asfurther described infra in the section “Cross-Competing Adnectins and/orAdnectins that Bind to the Same Adnectin Binding Site.” If the testAdnectin and reference molecule (e.g., another Adnectin or antibody)compete, then they bind to the same Adnectin binding site or to Adnectinbinding sites sufficiently proximal such that binding of one moleculeinterferes with the other.

The terms “specifically binds,” “specific binding,” “selective binding,and “selectively binds,” as used interchangeably herein refers to anAdnectin that exhibits affinity for a myostatin, but does notsignificantly bind (e.g., less than about 10% binding) to a differentpolypeptides as measured by a technique available in the art such as,but not limited to, Scatchard analysis and/or competitive binding assays(e.g., competition ELISA, BIACORE assay). The term is also applicablewhere e.g., a binding domain of an Adnectin of the invention is specificfor myostatin.

The term “preferentially binds” as used herein refers to the situationin which an Adnectin of the invention binds myostatin at least about 20%greater than it binds a different polypeptide as measured by a techniqueavailable in the art such as, but not limited to, Scatchard analysisand/or competitive binding assays (e.g., competition ELISA, BIACOREassay).

As used herein, the term “cross-reactivity” refers to an Adnectin whichbinds to more than one distinct protein having identical or very similarAdnectin binding sites.

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular Adnectin-protein(e.g., myostatin) interaction or the affinity of an Adnectin for aprotein (e.g., myostatin), as measured using a surface plasmon resonanceassay or a cell binding assay. A “desired K_(D),” as used herein, refersto a K_(D) of an Adnectin that is sufficient for the purposescontemplated. For example, a desired K_(D) may refer to the K_(D) of anAdnectin required to elicit a functional effect in an in vitro assay,e.g., a cell-based luciferase assay.

The term “k_(ass)”, as used herein, is intended to refer to theassociation rate constant for the association of an Adnectin into theAdnectin/protein complex.

The term “k_(diss)”, as used herein, is intended to refer to thedissociation rate constant for the dissociation of an Adnectin from theAdnectin/protein complex.

The term “IC₅₀”, as used herein, refers to the concentration of anAdnectin that inhibits a response, either in an in vitro or an in vivoassay, to a level that is 50% of the maximal inhibitory response, i.e.,halfway between the maximal inhibitory response and the untreatedresponse.

The term “myostatin activity” as used herein refers to one or more ofgrowth-regulatory or morphogenetic activities associated with thebinding of active myostatin protein to ActRIIb and the subsequentrecruitment of Alk4 or Alk5. For example, active myostatin is a negativeregulator of skeletal muscle mass. Active myostatin can also modulatethe production of muscle-specific enzymes (e.g., creatine kinase),stimulate myoblast proliferation, and modulate preadipocytedifferentiation to adipocytes. Myostatin activity can be determinedusing art-recognized methods, such as those described hererin.

The phrases “inhibit myostatin activity” or “antagonize myostatinactivity” or “antagonize myostatin” are used interchangeably to refer tothe ability of the anti-myostatin Adnectins of the present invention toneutralize or antagonize an activity of myostatin in vivo or in vitro.The terms “inhibit” or “neutralize” as used herein with respect to anactivity of an Adnectin of the invention means the ability tosubstantially antagonize, prohibit, prevent, restrain, slow, disrupt,eliminate, stop, reduce or reverse e.g., progression or severity of thatwhich is being inhibited including, but not limited to, a biologicalactivity or property, a disease or a condition. The inhibition orneutralization is preferably at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or higher.

For example, an anti-myostatin Adnectin of the invention may reducecirculating levels of biologically active myostatin normally found in avertebrate subject, or a reduction of circulating levels of biologicallyactive myostatin in subjects with disorders that result in elevatedcirculating levels of myostatin. A reduction of myostatin activity maybe determined using in vitro assays, e.g., binding assays, as describedherein. Alternatively, a reduction in myostatin activity may result inan increase in body weight, enhanced muscle mass, increased musclestrength, an alteration in the ratio of muscle to fat, an increase infat-free muscle mass, an increase in the size and/or number of musclecells, and/or a reduction in body fat content.

The term “PK” is an acronym for “pharmacokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” as used herein refers to any protein, peptide,or moiety that affects the pharmacokinetic properties of a biologicallyactive molecule when fused to or administered together with thebiologically active molecule. Examples of a PK modulation protein or PKmoiety include PEG, human serum albumin (HSA) binders (as disclosed inU.S. Publication Nos. 2005/0287153 and 2007/0003549, PCT PublicationNos. WO 2009/083804 and WO 2009/133208), human serum albumin, Fc or Fcfragments and variants thereof, and sugars (e.g., sialic acid).

The “half-life” of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to asubject a suitable dose of the amino acid sequence or compound of theinvention; collecting blood samples or other samples from the subject atregular intervals; determining the level or concentration of the aminoacid sequence or compound of the invention in said blood sample; andcalculating, from (a plot of) the data thus obtained, the time until thelevel or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is, for example, made to the standard handbooks, suchas Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinete Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

Half-life can be expressed using parameters such as the t_(1/2)-alpha,t_(1/2)-beta, HL_Lambda_z, and the area under the curve (AUC). In thepresent specification, an “increase in half-life” refers to an increasein any one of these parameters, any two of these parameters, any threeof these parameters or all four of these parameters. An “increase inhalf-life” in particular refers to an increase in the t_(1/2)-beta,and/or HL_Lambda_z, either with or without an increase in thet_(1/2)-alpha and/or the AUC or both.

The notations “mpk”, “mg/kg”, or “mg per kg” refer to milligrams perkilogram. All notations are used interchangeably throughout the presentdisclosure.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to an animal, preferably a mammalian (including anonprimate and a primate) or avian species, including, but not limitedto, murines, simians, humans, mammalian farm animals (e.g., bovine,porcine, ovine), mammalian sport animals (e.g., equine), and mammalianpets (e.g., canine and feline); preferably the term refers to humans.The term also refers to avian species, including, but not limited to,chickens and turkeys. In a certain embodiment, the subject, preferably amammal, preferably a human, is further characterized with a disease ordisorder or condition that would benefit from a decreased level ordecreased bioactivity of myostatin. In another embodiment the subject,preferably a mammal, preferably a human, is further characterized asbeing at risk of developing a disorder, disease or condition that wouldbenefit from a decreased level of myostatin or a decreased bioactivityof myostatin.

The term “therapeutically effective amount” refers to at least theminimal dose, but less than a toxic dose, of an agent which is necessaryto impart a therapeutic benefit to a subject. For example, atherapeutically effective amount of an anti-myostatin Adnectin of theinvention is an amount which in mammals, preferably humans, results inone or more of the following: an increase in muscle volume and/or musclestrength, a decrease in body fat, an increase in insulin sensitivity, orthe treatment of conditions wherein the presence of myostatin causes orcontributes to an undesirable pathological effect or a decrease inmyatostatin levels results in a beneficial therapeutic effect.

The term “frail” or “frailty” as used herein refers to a condition thatcan be characterized by two or more symptoms from weakness, weight loss,slowed mobility, fatigue, low activity levels, poor endurance, andimpaired behavioral response to sensory cues. One hallmark of frailty is“sarcopenia,” or the age-related loss of muscle mass.

The term “cachexia” as used herein refers to the condition ofaccelerated muscle wasting and loss of lean body mass that can resultfrom various diseases.

Overview

The present invention provides novel polypeptides that specifically bindto and antagonize myostatin (herein referred to as “anti-myostatinAdnectins”). In order to identify myostatin antagonists, myostatin waspresented to large synthetic libraries of Adnectins. Adnectins thatbound to myostatin were screened for myostatin binding, for biophysicalproperties, and for myostatin inhibitory activity. The anti-myostatinAdnectins were mutated and subjected to further selective pressure bylowering the target concentration and selecting for anti-myostatinAdnectins with slow off-rates. From this optimization process, a familyof Adnectins was identified as myostatin specific inhibitors withfavorable biochemical and biophysical activity. The anti-myostatinAdnectins disclosed in the present application are useful for thetreatment of disorders, diseases, and conditions for which inhibition ofmyostatin activity is known to be beneficial, including, but not limitedto, the treatment of muscle wasting diseases, metabolic disorders, andmuscle atrophy due to inactivity.

As disclosed in Rebbapragada et al. (MCB 2003; 23:7230-42), themyostatin signaling pathway involves binding of myostatin to ActRIIb,followed by recruitment of activin receptor-like kinase 4 (ALK4) orALK5. Binding to the ALKs induces Smad2/Smad3 phosphorylation, followedby activation of the TGFβ-like signaling pathway (see, e.g.,Rebbapragada et al., MCB 2003; 23:7230-42).

I. Fibronectin Based Scaffolds One aspect of the application providesanti-myostatin Adnectins comprising an Fn3 domain in which one or moreof the solvent accessible loops has been randomized or mutated. In someembodiments, the Fn3 domain is an Fn3 domain derived from the wild-typetenth module of the human fibronectin type III domain (¹⁰Fn3):

(SEQ ID NO: 4) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (BC,DE, and FG loops are underlined).

In other embodiments, the non-ligand binding sequences of ¹⁰Fn3, i.e.,the “¹⁰Fn3 scaffold”, may be altered provided that the ¹⁰Fn3 retainsligand binding function and/or structural stability. A variety of mutant¹⁰Fn3 scaffolds have been reported. In one aspect, one or more of Asp 7,Glu 9, and Asp 23 is replaced by another amino acid, such as, forexample, a non-negatively charged amino acid residue (e.g., Asn, Lys,etc.). These mutations have been reported to have the effect ofpromoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form (see, e.g., PCT Publication No. WO02/04523). A variety of additional alterations in the ¹⁰Fn3 scaffoldthat are either beneficial or neutral have been disclosed. See, forexample, Batori et al., Protein Eng., 15(12):1015-1020 (December 2002);Koide et al., Biochemistry, 40(34): 10326-10333 (Aug. 28, 2001).

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO:4, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues21-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87 (Xu et al.,Chemistry & Biology, 9:933-942, 2002).

Accordingly, in some embodiments, the anti-myostatin Adnectin is an¹⁰Fn3 polypeptide that is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%,85%, or 90% identical to the human ¹⁰Fn3 domain, shown in SEQ ID NO:4.Much of the variability will generally occur in one or more of theloops. Each of the beta or beta-like strands of a ¹⁰Fn3 polypeptide mayconsist essentially of an amino acid sequence that is at least 80%, 85%,90%, 95% or 100% identical to the sequence of a corresponding beta orbeta-like strand of SEQ ID NO:4, provided that such variation does notdisrupt the stability of the polypeptide in physiological conditions.

In some embodiments, the invention provides an anti-myostatin Adnectincomprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein the¹⁰Fn3 domain comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; aloop EF; and a loop FG; and has at least one loop selected from loop BC,DE, and FG with an altered amino acid sequence relative to the sequenceof the corresponding loop of the human ¹⁰Fn3 domain. In someembodiments, the anti-myostatin Adnectins of the present inventioncomprise an ¹⁰Fn3 domain comprising an amino acid sequence at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to the non-loop regions of SEQID NO:4, wherein at least one loop selected from BC, DE, and FG isaltered. In some embodiments, the BC and FG loops are altered, and insome embodiments, the BC, DE, and FG loops are altered, i.e., the ¹⁰Fn3domains comprise non-naturally occurring loops. In some embodiments, theAB, CD and/or the EF loops are altered. By “altered” is meant one ormore amino acid sequence alterations relative to a template sequence(corresponding human fibronectin domain) and includes amino acidadditions, deletions, substitutions or a combination thereof. Alteringan amino acid sequence may be accomplished through intentional, blind,or spontaneous sequence variation, generally of a nucleic acid codingsequence, and may occur by any technique, for example, PCR, error-pronePCR, or chemical DNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by 2-25 amino acids. In some embodiments, the length of theloop may be decreased by 1-11 amino acids. To optimize antigen binding,therefore, the length of a loop of ¹⁰Fn3 may be altered in length aswell as in sequence to obtain the greatest possible flexibility andaffinity in antigen binding.

In some embodiments, the polypeptide comprises a Fn3 domain thatcomprises an amino acid sequence at least 80, 85, 90, 95, 98, 99, or100% identical to the non-loop regions of SEQ ID NO: 4, wherein at leastone loop selected from BC, DE, and FG is altered. In some embodiments,the altered BC loop has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid substitutions, up to 1, 2, 3, or 4 amino acid deletions, up to 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, or a combinationthereof. In some embodiments, the altered DE loop has up to 1, 2, 3, 4,5, or 6 amino acid substitutions, up to 1, 2, 3, or 4 amino aciddeletions, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acidinsertions, or a combination thereof. In some embodiments, the FG loophas up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acidsubstitutions, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino aciddeletions, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid insertions, or acombination thereof.

The anti-myostatin Adnectins of the invention are based on an ¹⁰Fn3scaffold and are defined generally by the following sequence:

(SEQ ID NO: 5)EVVAAT(Z)_(a)SLLI(Z)_(x)YYRITYGE(Z)_(b)QEFTV(Z)_(y)ATI(Z)_(c)DYTITVYAV(Z)_(z)ISINYRT,wherein the AB loop is represented by (Z)_(a), the CD loop isrepresented by (Z)_(b), the EF loop is represented by (Z)_(e), the BCloop is represented by (Z)_(x), the DE loop is represented by (Z)_(y),and the FG loop is represented by (Z)_(z). Z represents any amino acidand the subscript following the Z represents an integer of the number ofamino acids. In particular, a may be anywhere from 1-15, 2-15, 1-10,2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; and b,c, x, y and z may each independently be anywhere from 2-20, 2-15, 2-10,2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7amino acids. In preferred embodiments, a is 2 amino acids, b is 7 aminoacids, c is 7 amino acids, x is 11 amino acids, y is 6 amino acids, andz is 12 amino acids. The sequences of the beta strands may have anywherefrom 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0to 3, from 0 to 2, or from 0 to 1 substitutions, deletions or additionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 4. In certain embodiments, the sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservativesubstitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 4. In certain embodiments,the core amino acid residues are fixed and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the core amino acid residues.

Alternatively, the anti-myostatin Adnectins of the invention are basedon an ¹⁰Fn3 scaffold and are defined generally by the sequence:

(SEQ ID NO: 6) EVVAATPTSLLI(Z)_(x)NYRITYGETGGNSPVQEFTV(Z)_(y)ATISGLKPGVDYTITVYAV(Z)_(z)ISINYRT wherein the BC loop is represented by (Z)_(x), the DE loop isrepresented by (Z)_(y), and the FG loop is represented by (Z)_(z). Zrepresents any amino acid and the subscript following the Z representsan integer of the number of amino acids. In particular, x, y and z mayeach independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15,5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. Inpreferred embodiments, x is 11 amino acids, y is 6 amino acids, and z is12 amino acids. The sequences of the beta strands may have anywhere from0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to3, from 0 to 2, or from 0 to 1 substitutions, deletions or additionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 1. In certain embodiments, the sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservativesubstitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 4. In certain embodiments,the core amino acid residues are fixed and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the core amino acid residues.

In certain embodiments, an anti-myostatin Adnectin described herein maycomprise the sequence as set forth in SEQ ID NO: 5 or 6, wherein atleast one of BC, DE, and FG loops as represented by (Z)_(x), (Z)_(y),and (Z)_(z), respectively, are altered. As described above, amino acidresidues corresponding to residues 21-30, 51-56, and 76-87 of SEQ ID NO:4 define the BC, DE, and FG loops, respectively. However, it should beunderstood that not every residue within the loop region needs to bemodified in order to achieve a ¹⁰Fn3 binder having strong affinity for adesired target (e.g., myostatin).

For example, residues 21 (S) and 22 (W) of the BC loop as shown in SEQID NO: 1 do not need to be modified for binding to myostatin. That is,¹⁰Fn3 domains with high affinity binding to myostatin may be obtained bymodifying only residues 23-30 of loop BC as shown in SEQ ID NO: 4. Thisis demonstrated in the BC loops exemplified in Table 1, which indicatesthat only the underlined positions are modified.

Similarly, positions 51 (P) and 56 (T) of loop DE as shown in SEQ ID NO:4 do not need to be modified for binding myostatin. That is, ¹⁰Fn3domains with high affinity binding to myostatin may be obtained bymodifying only residues 52-55 of loop DE as shown in SEQ ID NO: 4. Thisis demonstrated in the DE loops exemplified in Table 1, which indicatesthat only resides spanning the underlined positions were altered.

Likewise, positions 76 (T) and 87 (P) of the FG loop as shown in SEQ IDNO: 1 do not need to be modified for binding myostatin. That is, ¹⁰Fn3domains with high affinity binding to myostatin may be obtained bymodifying only residues 77-86 of loop FG as shown in SEQ ID NO: 4. Thisis demonstrated in the FG loops exemplified in Table 1, which indicatesthat only the residues spanning the underlined positions were altered.

Accordingly, in some embodiments, the BC, DE, and FG loop regions of theanti-myostatin Adnectins of the invention can be described according toconsensus sequences. These consensus sequences are exemplified by theBC, DE, and FG loops shown in Table 1, and as determined by WebLogoanalysis (FIGS. 2-7) (Crooks G E, Hon G, Chandonia J M, Brenner S E.WebLogo: A sequence logo generator. Genome Research 2004; 14:1188-1190,herein incorporated by reference in its entirety). WebLogo analysisgenerates an amino acid signature reflecting the frequency of aminoacids in each altered position of the BC, DE, or FG loop.

For example, in some embodiments, the BC loop, (Z), is defined by theconsensus sequence X₁-L-P-X₂-X₃-X₄-X₅-X₆-X₇, wherein, X₁ is S, T or Y;X₂ is H, Y, N, R, F, G, S or T; X₃ is A, P, Q, S, F, H, N or R; X₄ is Gor A; X₅ H, L, R, V, N, D, F, I or K; X₆ is A, L, G, M, F, I or V; andX₇ is H or N. In certain preferred embodiments, the BC loop comprises anamino acid sequence selected from SEQ ID NOs: 7, 11-21, 23-31, 34, and36-38. In one embodiment, the BC loop comprises the amino acid set forthin SEQ ID NO. 34.

In some embodiments, the DE loop, (Z)_(y), is defined by the consensussequence G-R-G-X₈, wherein X₈ is V or L. In certain preferredembodiments, the DE loop comprises an amino acid selected from SEQ IDNOs: 39 and 42. In one embodiment, the DE loop comprises the amino acidset forth in SEQ ID NO. 39.

In some embodiments, the FG loop, (Z)_(z), is defined by consensussequence X₉-X₁₀-X₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈, wherein X₉ is L, V or I;X₁₀ is T or S; X₁₁ is K, R, A, G, S, D, H, N, T or P; X₁₂ is S, T, A, E,H, K or N; X₁₃ is K, G, Q, D, E, N, T or S; X₁₄ is V, I, F, L, M, P, Tor Y; X₁₅ is I, L or Y; X₁₆ is H, I, V, K, L, R, F, G, S or T; X₁₇ is Yor H; and X₁₈ is K, M, L, R or V. In certain preferred embodiments, theFG loop comprises an amino acid sequence selected from SEQ ID NOs: 46,50-62, 64-72, 75-77, and 79. In one embodiment, the FG loop comprisesthe amino acid set forth in SEQ ID NO. 75.

In other embodiments, the BC loop, (Z)_(x), is defined by the consensussequence X₁₉-X₂₀-P-X₂₁-G-X₂₂-A, wherein X₁₉ is D, E, V or W; X₂₀ is A, Sor V; X₂₁ is R, A, G, K or L; and X₂₂ is L or R. In certain preferredembodiments, the BC loop comprises an amino acid sequence selected fromSEQ ID NOs: 8-10, 22, 32, 33, and 35.

In other embodiments, the DE loop, (Z)_(y), is defined by the consensussequence X₂₃-G-R-G-X₂₄, wherein X₂₃ is V, P, F, I or L; and X₂₄ is S, Nor T. In certain preferred embodiments, the DE loop comprises and aminoacid sequence selected from SEQ ID NOs: 40, 41, and 43-45.

In other embodiments, the FG loop, (Z)_(z), is defined by the consensussequence X₂₅-X₂₆-R-X₂₇-G-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂, wherein X₂₅ is I or V; X₂₆is F, D or Y; X₂₇ is D or T; X₂₈ is P, M, V or T; X₂₉ is V, L, N, R orS; X₃₀ is H, T, L, N, Q or S; X₃₁ is F, W, Y, H or L; and X₃₂ is D, A orG. In certain preferred embodiments the FG loop comprises an amino acidsequence selected from SEQ ID NOs: 47-49, 63, 73, 74, and 78.

Accordingly, in certain embodiments the invention provides ananti-myostatin Adnectin comprising a BC loop, (Z)_(x), having thesequence X₁-L-P-X₂-X₃-X₄-X₅-X₆-X₇ and a DE loop, (Z)_(y), having thesequence G-R-G-X₈, as defined above. In certain embodiments, the BC loopcomprises an amino acid sequence selected from SEQ ID NOs: 7, 11-21,23-31, 34, and 36-38, and the DE loop comprises an amino acid sequenceselected from SEQ ID NOs: 39 and 42. In one embodiment, the BC and DEloops comprise the amino acid sequences set forth in SEQ ID NOs: 34 and39, respectively.

In certain embodiments, the anti-myostatin Adnectin comprises a BC loop,(Z)_(x), having the sequence X₁-L-P-X₂-X₃-X₄-X₅-X₆-X₇ and an FG loop,(Z)_(z), having the sequence X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈, asdefined above. In certain embodiments, the BC loop comprises an aminoacid sequence selected from SEQ ID NOs: 7, 11-21, 23-31, 34, and 36-38,and the FG loop comprises an amino acid sequence selected from SEQ IDNOs: 46, 50-62, 64-72, 75-77, and 79. In one embodiment, the BC and FGloops comprise the amino acid sequences set forth in SEQ ID NOs: 34 and75, respectively.

In certain embodiments the anti-myostatin Adnectin comprises a DE loop,(Z)_(y), having the sequence G-R-G-X₈ and an FG loop, (Z)_(z), havingthe sequence X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈, as defined above.In certain embodiments, the DE loop comprises an amino acid sequenceselected from SEQ ID NOs: 39 and 42, and the FG loop comprises an aminoacid sequence selected from SEQ ID NOs: 46, 50-62, 64-72, 75-77, and 79.In one embodiment, the DE and FG loops comprise the amino acid sequencesset forth in SEQ ID NOs: 39 and 75, respectively.

In certain embodiments, the anti-myostatin Adnectin comprises a BC loop,(Z)_(x), having the sequence X₁-L-P-X₂-X₃-X₄-X₅-X₆-X₇, a DE loop,(Z)_(y), having the sequence G-R-G-X₈ and an FG loop, (Z)_(z), havingthe sequence X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈, as defined above.In certain embodiments, the BC loop comprises an amino acid sequenceselected from SEQ ID NOs: 7, 11-21, 23-31, 34, and 36-38, the DE loopcomprises an amino acid sequence selected from SEQ ID NOs: 39 and 42,and the FG loop comprises an amino acid sequence selected from SEQ IDNOs: 46, 50-62, 64-72, 75-77, and 79. In one embodiment, the BC, DE, andFG loops comprise the amino acid sequences set forth in SEQ ID NOs: 34,39, and 75, respectively.

In other embodiments the invention provides an anti-myostatin Adnectincomprising a BC loop, (Z)_(x), having the sequence X₁₉-X₂₀-P-X₂₁-G-X₂₂-Aand a DE loop, (Z)_(y), having the sequence X₂₃-G-R-G-X₂₄, as definedabove. In certain embodiments, the BC loop comprises an amino acidsequence selected from SEQ ID NOs: 8-10, 22, 32, 33, and 35 and the DEloop comprises an amino acid sequence selected from SEQ ID NOs: 40, 41,and 43-45.

In other embodiments, the anti-myostatin Adnectin comprises a BC loop,(Z)_(x), having the sequence X₁₉-X₂₀-P-X₂₁-G-X₂₂-A and an FG loop,(Z)_(z), having the sequence X₂₅-X₂₆-R-X₂₇-G-X₂₈-X₂₉-X₃₀-X₃₀-X₃₂, asdefined above. In certain embodiments, the BC loop comprises an aminoacid sequence selected from SEQ ID NOs: 8-10, 22, 32, 33, and 35 and theFG loop comprises an amino acid sequence selected from SEQ ID NOs:47-49, 63, 73, 74, and 78.

In other embodiments the anti-myostatin Adnectin comprises a DE loop,(Z)_(y), having the sequence X₂₃-G-R-G-X₂₄ and an FG loop, (Z)_(z),having the sequence X₂₅-X₂₆-R-X₂₇-G-X₂₈-X₂₉-X₃₀-X₃₀-X₃₂, as definedabove. In certain embodiments, the DE loop comprises an amino acidsequence selected from SEQ ID NOs: 40, 41, and 43-45 and the FG loopcomprises an amino acid sequence selected from SEQ ID NOs: 47-49, 63,73, 74, and 78.

In other embodiments the anti-myostatin Adnectin comprises a BC loop,(Z)_(x), having the sequence X₁₉-X₂₀-P-X₂₁-G-X₂₂-A, comprises a DE loop,(Z)_(y), having the sequence X₂₃-G-R-G-X₂₄ and an FG loop, (Z)_(z),having the sequence X₂₅-X₂₆-R-X₂₇-G-X₂₈-X₂₉-X₃₀-X₃₀-X₃₂, as definedabove. In certain embodiments, the BC loop comprises an amino acidsequence selected from SEQ ID NOs: 8-10, 22, 32, 33, and 35, the DE loopcomprises an amino acid sequence selected from SEQ ID NOs: 40, 41, and43-45, and the FG loop comprises an amino acid sequence selected fromSEQ ID NOs: 47-49, 63, 73, 74, and 78.

In certain preferred embodiments, an anti-myostatin Adnectin of theinvention comprises the sequence set forth in SEQ ID NO: 5 or 6, whereinBC, DE and FG loops as represented by (Z)_(x), (Z)_(y), and (Z)_(z),respectively, are replaced with a respective set of BC, DE, and FG loopshaving the consensus sequences of SEQ ID NOs: 7-38, 39-45, and 46-79,respectively.

In other preferred embodiments, an anti-myostatin Adnectin of theinvention comprises the sequence set forth in SEQ ID NO: 5 or 6, whereinBC, DE and FG loops as represented by (Z)_(x), (Z)_(y), and (Z)_(z),respectively, are replaced with a respective set of BC, DE, and FG loopshaving sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences of the clones listed inTable 1.

In exemplary embodiments, an anti-myostatin Adnectin as described hereinis defined by SEQ ID NO: 5 and has a respective set of BC, DE and FGloop sequences from any of the clones listed in Table 1. For example,clone 1979_B06 in Table 1 comprises BC, DE, and FG loops as set forth inSEQ ID NOs: 7, 39, and 46, respectively. Therefore, an anti-myostatinAdnectin based on these loops may comprise SEQ ID NO: 5 or 6, wherein(Z)_(x) comprises SEQ ID NO: 7, (Z)_(y) comprises SEQ ID NO: 39, and(Z)_(z) comprises SEQ ID NO: 46. Similar constructs are contemplatedutilizing the set of BC, DE and FG loops from the other clones in Table1, or the consensus sequences of SEQ ID NOs: 7-38, 39-45, and 46-79,respectively. The scaffold regions of such anti-myostatin Adnectins maycomprise anywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8,from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from0 to 1 substitutions, conservative substitutions, deletions or additionsrelative to the scaffold amino acids residues of SEQ ID NO: 4. Suchscaffold modifications may be made, so long as the anti-myostatinAdnectin is capable of binding myostatin with a desired K_(D).

In preferred embodiments, the BC loop of the anti-myostatin Adnectin ofthe invention comprises an amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 7) SWSLPHAGHVN,  (SEQ ID NO: 8) SWVSPRGRAR, (SEQ ID NO: 9)SWEVPRGLAR,  (SEQ ID NO: 10) SWWAPLGLAR, (SEQ ID NO: 11) SWTLPHAGLAH, (SEQ ID NO: 12) SWYLPYPAHMN, (SEQ ID NO: 13) SWSLPFAGHLN, (SEQ ID NO: 14) SWSLPYSGLAN, (SEQ ID NO: 15) SWSLPHAGHAH, (SEQ ID NO: 16) SWTLPNFGLIN, (SEQ ID NO: 17) SWTLPHAGRAH, (SEQ ID NO: 18) SWSLPYAGHLN, (SEQ ID NO: 19) SWSLPYAAHMN, (SEQ ID NO: 20) SWSLPYPGHLN, (SEQ ID NO: 21) SWSLPYAGHAH, (SEQ ID NO: 22) SWDAPGGLAR, (SEQ ID NO: 23) SWSLPTPGLAH, (SEQ ID NO: 24) SWSLPHRGVAN, (SEQ ID NO: 25) SWSLPSSGVAH, (SEQ ID NO: 26) SWSLPHHGFGH, (SEQ ID NO: 27) SWSLPHAGDAH, (SEQ ID NO: 28) SWSLPHNGVAH, (SEQ ID NO: 29) SWSLPRQGLAN, (SEQ ID NO: 30) SWSLPGPGHFH, (SEQ ID NO: 31) SWSLPHPGLGH, (SEQ ID NO: 32) SWDAPRGLAR, (SEQ ID NO: 33) SWDAPAGLAR,  (SEQ ID NO: 34)SWSLPHQGKAN, (SEQ ID NO: 35) SWDAPKGLAR,  (SEQ ID NO: 36) SWSLPNPGIAH,(SEQ ID NO: 37) SWSLPRPGNAH,  and (SEQ ID NO: 38) SWSLPNPGNAH.In some embodiments, the BC loop of the anti-myostatin Adnectin of theinvention comprises the underlined portion of any one of SEQ ID NOs:7-38, as shown in Table 1. In one embodiment, the BC loop comprises theunderlined portion of SEQ ID NO: 34.

In some embodiments, the DE loop of the anti-myostatin Adnectin of theinvention comprises an amino acid sequence selected from the groupconsisting of: PGRGVT (SEQ ID NO: 39), PGRGST (SEQ ID NO: 40), LGRGST(SEQ ID NO: 41), PGRGLT (SEQ ID NO: 42), IGRGST (SEQ ID NO: 43), FGRGTT(SEQ ID NO: 44), and VGRGNT (SEQ ID NO: 45). In some embodiments, the DEloop of the anti-myostatin Adnectin of the invention comprises theunderlined portion of any one of SEQ ID NOs: 39-45, as shown in Table 1.In one embodiment, the DE loop comprises the underlined portion of SEQID NO: 39.

In some embodiments, the FG loop of anti-myostatin Adnectin of theinvention comprises an amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 46) TLTKSQMIHYMP,  (SEQ ID NO: 47) TIYRDGMSHHDP,(SEQ ID NO: 48) TVYRDGPLLLAP,  (SEQ ID NO: 49) TIFRTGMVQYDP,(SEQ ID NO: 50) TLTNSEIILYKP,  (SEQ ID NO: 51) TLTKSQILHHRP,(SEQ ID NO: 52) TLTRSKIIHYMP,  (SEQ ID NO: 53) TLTHSNIIRYVP,(SEQ ID NO: 54) TVSSTKVIVYLP,  (SEQ ID NO: 55) TITKSTIIIYKP,(SEQ ID NO: 56) TVTTTSVILYKP,  (SEQ ID NO: 57) TLTKSQLIHYMP,(SEQ ID NO: 58) TLTRSQVIHYMP,  (SEQ ID NO: 59) TLTKSKIIHYMP,(SEQ ID NO: 60) TVSSTKVIHYKP,  (SEQ ID NO: 61) TLTKSKVIHYMP,(SEQ ID NO: 62) TVTTTKVIHYKP,  (SEQ ID NO: 63) TIDRDGVNHFAP,(SEQ ID NO: 64) TVTHHGVIGYKP,  (SEQ ID NO: 35) TLTGANVIIYKP,(SEQ ID NO: 66) TVTNTGVIIYKP,  (SEQ ID NO: 67) TVTATGIIIYKP,(SEQ ID NO: 68) TVTRAGFYRYKP,  (SEQ ID NO: 69) TVTREEVISYKP,(SEQ ID NO: 70) TVTAAGVIIYKP,  (SEQ ID NO: 71) TVTANQPIIYKP,(SEQ ID NO: 72) TITPETIIVYKP,  (SEQ ID NO: 73) TIDRDGTRSFDP,(SEQ ID NO: 74) TIFRDGPVTWDP,  (SEQ ID NO: 75) TVTDTGYLKYKP,(SEQ ID NO: 76) TLTGSDTIFYKP,  (SEQ ID NO: 77) TVTGKDVIKYKP,(SEQ ID NO: 78) TIFRDGVVNYGP,  and  (SEQ ID NO: 79) TVTDTGFITYKP.In some embodiments, the FG loop of the anti-myostatin Adnectin of theinvention comprises the underlined portion of any one of SEQ ID NOs:46-79, as shown in Table 1. In one embodiment, the FG loop comprises theunderlined portion of SEQ ID NO: 75.

In some embodiments, the anti-myostatin Adnectin of the inventioncomprises one BC loop sequence selected from the BC loop sequenceshaving SEQ ID NOs: 7-38, or the underlined portion of any one of SEQ IDNOs: 7-38, as shown in Table 1; one DE loop sequence selected from theDE loop sequences having SEQ ID NOs: 39-45, or the underlined portion ofany one of SEQ ID NOs: 39-45 as shown in Table 1; and one FG loopsequence selected from the FG loop sequences having SEQ ID NOS: 46-79,or the underlined portion of any one of SEQ ID NOS: 46-79 as shown inTable 1. In some embodiments, the anti-myostatin Adnectin of theinvention comprises a BC, DE and FG loop amino acid sequence at least70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to of any oneof SEQ ID NOs: 7-38, 39-45, and 46-79, respectively. In otherembodiments, the anti-myostatin Adnectin of the invention comprises aBC, DE and FG loop amino acid sequence at least 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% or 100% identical to the underlined portion of any one ofSEQ ID NOS: 7-38, 39-45, and 46-79, respectively, as shown in Table 1.

In some embodiments, the anti-myostatin Adnectin comprises the aminoacid sequence of any one of SEQ ID NOs: 80-123, 228-239, and 252-273(full length sequences from Tables 2, 5, and 6). In one embodiment, theanti-myostatin Adnectin comprises the amino acid sequence of SEQ ID NO:273.

In some embodiments, the anti-myostatin Adnectin comprises an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to any one of SEQ ID NOS: 80-123, 228-239, and 252-273. Inother embodiments, the anti-myostatin Adnectins comprise an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to thenon-BC, DE, and FG loop regions of SEQ ID NOs: 80-123, 228-239, and252-273.

In one embodiment, the anti-myostatin Adnectin of the inventioncomprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 34, 39,and 75, respectively. In another embodiment, the anti-myostatin Adnectincomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence set forth in SEQID NO: 273 [PRD-1474], SEQ ID NO: 118 [3116_A06], or SEQ ID NO: 281[core Adnectin sequence shared by PRD-1474 and 3116_A06, preceded by aN-terminal extension sequence (GVSDVPRDL) and followed by a C-terminaltail (EI)]. In yet another embodiment, the anti-myostatin Adnectin ofthe present invention comprises an amino acid sequence at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loopregions of SEQ ID NO: 273, 118, or 281.

In some embodiments, the anti-myostatin Adnectin of the inventioncomprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 34, 39,and 75, respectively, wherein the BC loop comprises 1, 2, 3, 4, 5, or 6amino acid substitutions, such as conservative amino acid substitutions.Accordingly, in some embodiments, the BC loop is defined by theconsensus sequence X₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T orY; X₃₄ is Y, N, R, F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ isA; X₃₇ is H, L, R, V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉is H.

In some embodiments, the anti-myostatin Adnectin of the inventioncomprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 34, 39,and 75, respectively, wherein the DE loop comprises 1 amino acidsubstitution, such as a conservative amino acid substitution.Accordingly, in some embodiments, the DE loop is defined by theconsensus sequence G-R-G-X₄₀, wherein X₄₀ is L.

In some embodiments, the anti-myostatin Adnectin of the inventioncomprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 34, 39,and 75, respectively wherein the FG loop comprises 1, 2, 3, 4, 5, 6, 7,or 8 amino acid substitutions, such as conservative amino acidsubstitutions. Accordingly, in some embodiments, the FG loop is definedby the consensus sequence X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀,wherein X₄₁ is L or I; X₄₂ is S; X₄₃ is K, R, A, G, S, H, N, T, or P;X₄₄ is S, A, E, H, K, or N; X₄₅ is K, Q, D, E, N, T, or S; X₄₆ is V, I,F, L, M, P, or T; X₄₇ is I or Y; X₄₈ is H, I, V, L, R, F, G, S, or T;X₄₉ is H; and X₅₀ is M, L, R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H, and the DEloop has an amino acid sequence according to the formula G-R-G-X₄₀,wherein X₄₀ is L.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H, and the FGloop has an amino acid sequence according to the formulaX₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I; X₄₂ isS; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, or N; X₄₅is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ is I or Y;X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L, R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the DE loophas an amino acid sequence according to the formula G-R-G-X₄₀, whereinX₄₀ is L, and the FG loop has an amino acid sequence according to theformula X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I;X₄₂ is S; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, orN; X₄₅ is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ isI or Y; X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L,R, or V.

In some embodiments, the polypeptide has the BC, DE, and FG loops as setforth in SEQ ID NOs: 34, 39, and 75, respectively, wherein the BC loophas an amino acid sequence according to the formulaX₃₃-L-P-X₃₄-X₃₅-X₃₆-X₃₇-X₃₈-X₃₉, wherein X₃₃ is T or Y; X₃₄ is Y, N, R,F, G, S, or T; X₃₅ is A, P, S, F, H, N, or R; X₃₆ is A; X₃₇ is H, L, R,V, N, D, F, or I; X₃₈ is L, G, M, F, I, or V; and X₃₉ is H; the DE loophas an amino acid sequence according to the formula G-R-G-X₄₀, whereinX₄₀ is L; and the FG loop has an amino acid sequence according to theformula X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈-X₄₉-X₅₀, wherein X₄₁ is L or I;X₄₂ is S; X₄₃ is K, R, A, G, S, H, N, T, or P; X₄₄ is S, A, E, H, K, orN; X₄₅ is K, Q, D, E, N, T, or S; X₄₆ is V, I, F, L, M, P, or T; X₄₇ isI or Y; X₄₈ is H, I, V, L, R, F, G, S, or T; X₄₉ is H; and X₅₀ is M, L,R, or V.

In some embodiments, the anti-myostatin Adnectin is encoded by a nucleicacid sequence as set forth in any one of SEQ ID NOs: 124-167, 240-251,and 284-305 (full length sequences from Tables 2, 5, and 6). In someembodiments, the anti-myostatin Adnectin is encoded by a nucleic acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to any one of SEQ ID NOS: 124-167, 240-251, and 284-305.

Fibronectin naturally binds certain types of integrins through itsintegrin-binding motif, “arginine-glycine-aspartic acid” (RGD). In someembodiments, the polypeptide comprises a ¹⁰Fn3 domain that lacks the(RGD) integrin binding motif. The integrin binding domain may be removedby altering the RGD sequence by amino acid substitution, deletion orinsertion.

In some embodiments, BC, DE and/or FG loop amino acid sequencesidentical to the underlined portion of any one of SEQ ID NOS: 7-38,39-45, and 46-79, respectively, as shown in Table 1, are grafted intonon-¹⁰Fn3 domain protein scaffolds. For instance, one or more loop aminoacid sequences is exchanged for or inserted into one or more CDR loopsof an antibody heavy or light chain or fragment thereof. In otherembodiments, the protein domain into which one or more amino acid loopsequences are exchanged or inserted includes, but is not limited to,consensus Fn3 domains (Centocor, US), ankyrin repeat proteins (MolecularPartners AG, Zurich Switzerland), domain antibodies (Domantis, Ltd,Cambridge, Mass.), single domain camelid nanobodies (Ablynx, Belgium),Lipocalins (e.g., anticalins; Pieris Proteolab AG, Freising, Germany),Avimers (Amgen, Calif.), affibodies (Affibody AG, Sweden), ubiquitin(e.g., affilins; Scil Proteins GmbH, Halle, Germany), protein epitopemimetics (Polyphor Ltd, Allschwil, Switzerland), helical bundlescaffolds (e.g. alphabodies, Complix, Belgium), Fyn SH3 domains (CovagenAG, Switzerland), or atrimers (Anaphor, Inc., CA).

The SEQ ID NOs of the BC, DE and FG loops of the exemplaryanti-myostatin Adnectins of the invention are presented in Table 1.

TABLE 1 Anti-Myostatin Adnectin BC, DE, and FG Loops Loop SequencesSEQ ID SEQ ID SEQ ID Clone BC Loop NO: DE Loop NO: FG Loop NO: 1979_B06SWSLPHAGHVN  7 PGRGVT 39 TLTKSQMIHYMP 46 2062_G02 SWVSPRGRAR  8 PGRGST40 TIYRDGMSHHDP 47 2522_C09 SWEVPRGLAR  9 LGRGST 41 TVYRDGPLLLAP 482523_G06 SWWAPLGLAR 10 PGRGST 40 TIFRTGMVQYDP 49 2524_C11 SWTLPHAGLAH 11PGRGVT 39 TLTNSEIILYKP 50 2524_D09 SWYLPYPAHMN 12 PGRGLT 42 TLTKSQILHHRP51 2524_E10 SWSLPFAGHLN 13 PGRGVT 39 TLTRSKIIHYMP 52 2524_H05SWSLPYSGLAN 14 PGRGVT 39 TLTHSNIIRYVP 53 2524_H11 SWSLPHAGHAH 15 PGRGVT39 TVSSTKVIVYLP 54 2525_B01 SWTLPNFGLIN 16 PGRGVT 39 TITKSTIIIYKP 552525_D02 SWTLPHAGRAH 17 PGRGVT 39 TVTTTSVILYKP 56 2525_D05 SWSLPYAGHLN18 PGRGVT 39 TLTKSQLIHYMP 57 2525_F07 SWSLPYAAHMN 19 PGRGVT 39TLTRSQVIHYMP 58 2987_A06 SWSLPHAGHAH 15 PGRGVT 39 TLTKSKIIHYMP 592987_B04 SWSLPYPGHLN 20 PGRGVT 39 TLTKSKIIHYMP 59 2987_B09 SWTLPHAGRAH17 PGRGVT 39 TLTRSKIIHYMP 52 2987_C02 SWSLPYAGHAH 21 PGRGVT 39TLTKSKIIHYMP 59 2987_D05 SWSLPHAGHAH 15 PGRGVT 39 TLTRSKIIHYMP 522987_E03 SWSLPYPGHLN 20 PGRGVT 39 TLTRSKIIHYMP 52 2987_E08 SWTLPHAGRAH17 PGRGVT 39 TVSSTKVIHYKP 60 2987_F01 SWSLPYAGHAH 21 PGRGVT 39TLTRSKIIHYMP 52 2987_F06 SWSLPHAGHAH 15 PGRGVT 39 TLTKSKVIHYMP 612987_G04 SWSLPYPGHLN 20 PGRGVT 39 TLTKSKVIHYMP 61 2987_G09 SWTLPHAGRAH17 PGRGVT 39 TVSSTKVIVYLP 54 2987_H02 SWSLPYAGHAH 21 PGRGVT 39TLTKSKVIHYMP 61 2987_H07 SWTLPHAGRAH 17 PGRGVT 39 TVTTTKVIHYKP 623006_A10 SWDAPGGLAR 22 IGRGST 43 TIDRDGVNHFAP 63 3007_B08 SWSLPTPGLAH 23PGRGVT 39 TVTHHGVIGYKP 64 3007_C09 SWSLPHRGVAN 24 PGRGVT 39 TLTGANVIIYKP65 3007_C10 SWSLPSSGVAH 25 PGRGVT 39 TVTNTGVIIYKP 66 3008_A03SWSLPHHGFGH 26 PGRGVT 39 TVTATGIIIYKP 67 3008_B08 SWSLPHAGDAH 27 PGRGVT39 TVTRAGFYRYKP 68 3008_D04 SWSLPHNGVAH 28 PGRGVT 39 TVTREEVISYKP 693008_F01 SWSLPRQGLAN 29 PGRGVT 39 TVTAAGVIIYKP 70 3008_G01 SWSLPGPGHFH30 PGRGVT 39 TVTANQPIIYKP 71 3008_G03 SWSLPHPGLGH 31 PGRGVT 39TITPETIIVYKP 72 3115_D04 SWDAPRGLAR 32 FGRGTT 44 TIDRDGTRSFDP 733115_E06 SWDAPAGLAR 33 VGRGNT 45 TIFRDGPVTWDP 74 3116_A06 SWSLPHQGKAN 34PGRGVT 39 TVTDTGYLKYKP 75 3116_A07 SWDAPKGLAR 35 VGRGNT 45 TIFRDGPVTWDP74 3116_C01 SWSLPNPGIAH 36 PGRGVT 39 TLTGSDTIFYKP 76 3116_C06SWSLPRPGNAH 37 PGRGVT 39 TVTGKDVIKYKP 77 3116_H06 SWDAPAGLAR 33 VGRGNT45 TIFRDGVVNYGP 78 3146_A08 SWSLPNPGNAH 38 PGRGVT 39 TVTDTGFITYKP 79

The SEQ ID NOs of exemplary anti-myostatin monoAdnectins of theinvention are presented in Table 2.

TABLE 2 Anti-Myostatin MonoAdnectins Sequence Amino Acid SequenceNucleic Acid Sequence 1979_B06 also MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT referred to herein asWSLPHAGHVNYYRITYGETGGN GGAAGTGGTTGCTGCCACCCCCACCAGCC ATI-1133SPVQEFTVPGRGVTATISGLKPG TGCTGATCAGCTGGTCTCTGCCGCATGCT (Adnectin core 1VDYTITVYAVTLTKSQMIHYMPI GGTCATGTGAACTATTACCGCATCACTTA sequence havingSINYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTC AdNT1 (underlined)ID NO: 80) CAGGAGTTCACTGTGCCTGGTCGTGGTGT and AdCT1 (italics)TACAGCTACCATCAGCGGCCTTAAACCTG terminal sequenceGCGTTGATTATACCATCACTGTGTATGCT with His6 tag)GTCACTCTGACTAAATCTCAGATGATCCA TTACATGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATC ACCACCACCAC (SEQ ID NO: 124) 2062_G02 alsoMGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCTreferred to herein as WVSPRGRARYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC ATI-1134 PVQEFTVPGRGSTATISGLKPGVTGCTGATCAGCTGGGTTTCTCCGCGTGGT (Adnectin core 2 DYTITVYAVTIYRDGMSHHDPISICGTGCTCGATATTACCGCATCACTTACGG sequence having NYRTEIDKPSQHHHHHH (SEQCGAAACAGGAGGCAATAGCCCTGTCCAG AdNT1 (underlined) ID NO: 81)GAGTTCACTGTGCCTGGTCGTGGTTCTAC and AdCT1 (italics)AGCTACCATCAGCGGCCTTAAACCTGGCG terminal sequenceTTGATTATACCATCACTGTGTATGCTGTCA with His6 tag)CTATCTACCGTGACGGTATGTCTCATCAT GACCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACC ACCACCAC (SEQ ID NO: 125) 2522_C09MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 3WEVPRGLARYYRITYGETGGNS GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingPVQEFTVLGRGSTATISGLKPGV TGCTGATCAGCTGGGAAGTGCCGCGTGGC AdNT1 (underlined)DYTITVYAVTVYRDGPLLLAPISI CTAGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 82) GAGTTCACTGTGCTTGGTCGTGGTTCTACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTGTGTACCGTGACGGGCCGTTGCTTCTTGCCCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 126) 2523_G06 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 4 WWAPLGLARYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVPGRGSTATISGLKPGVTGCTGATCAGCTGGTGGGCCCCGCTGGGT AdNT1 (underlined)DYTITVYAVTIFRTGMVQYDPISI CTTGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 83) GAGTTCACTGTGCCTGGTCGGGGCTCTACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTACGGGCATGGTTCAATATGACCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 127) 2524_C11 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 5 WTLPHAGLAHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGACTCTGCCGCATGCT AdNT1 (underlined)VDYTITVYAVTLTNSEIILYKPISI GGTCTTGCGCACTATTACCGCATCACTTAand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 84) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTCTGACTAATTCTGAGATTATCCTTTACAAGCCAATTTCCATTAATTACCGCAC AGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 128) 2524_D09 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 6 WYLPYPAHMNYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGLTATISGLKPGTGCTGATCAGCTGGTACCTCCCGTATCCT AdNT1 (underlined)VDYTITVYAVTLTKSQILHHRPIS GCGCATATGAACTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 85) CAGGAGTTCACTGTGCCTGGGCGGGGTCTwith His6 tag) GACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTCTGACAAAATCTCAGATTCTCCATCATAGGCCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 129) 2524_E10 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 7 WSLPFAGHLNYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCATTGCCGTTTGCTG AdNT1 (underlined)VDYTITVYAVTLTRSKIIHYMPIS GTCATTTGAACTATTACCGCATCACTTACand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence ID NO: 86) AGGAGTTCACTGTGCCTGGTCGTGGTGTTwith His6 tag) ACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGT CACTCTGACTCGCTCTAAGATTATTCATTATATGCCAATTTCCATTAATTACCGCACAG AAATTGACAAACCATCCCAGCACCATCACCACCACCAC(SEQ ID NO: 130) 2524_H05 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 8 WSLPYSGLANYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCTTATTCTG AdNT1 (underlined)VDYTITVYAVTLTHSNIIRYVPISI GCCTTGCGAACTATTACCGCATCACTTACand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence ID NO: 87) AGGAGTTCACTGTGCCTGGTCGTGGGGTTwith His6 tag) ACAGCTACTATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGT CACTCTGACTCACTCTAATATAATTCGATACGTGCCAATTTCCATTAATTACCGCACA GAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 131) 2524_H11 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 9 WSLPHAGHAHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCCCTACCGCATGCG AdNT1 (underlined)VDYTITVYAVTVSSTKVIVYLPIS GGTCATGCGCACTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 88) CAGGAGTTCACTGTGCCTGGTCGTGGAGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTGTCTAGTACAAAGGTGATAGTTTACCTGCCAATTTCCATTAATTACCGCAC AGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 132) 2525_B01 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 10 WTLPNFGLINYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVPGRGVTATISGLKPGVTGCTGATCAGCTGGACTTTGCCGAATTTC AdNT1 (underlined)DYTITVYAVTITKSTIIIYKPISINY GGTCTTATTAATTATTACCGCATCACTTACand AdCT1 (italics) RTEIDKPSQHHHHHH (SEQ ID GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence NO: 89) AGGAGTTCACTGTGCCTGGTCGTGGTGTT with His6 tag)ACAGCTACCATCAGCGGCCTTAAACCTGG CGTTGATTATACCATCACTGTGTATGCTGTCACTATCACCAAATCTACTATCATCATTTA CAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCAC CACCACCAC (SEQ ID NO: 133) 2525_D02MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 11WTLPHAGRAHYYRITYGETGGN GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATCAGCTGGACTTTGCCGCATGCT AdNT1 (underlined)VDYTITVYAVTVTTTSVILYKPIS GGTCGTGCGCACTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 90) CAGGAGTTCACTGTGCCTGGGCGGGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTGACGACAACTTCGGTGATCCTTTACAAGCCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 134) 2525_D05 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 12 WSLPYAGHLNYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTTCCTTATGCTG AdNT1 (underlined)VDYTITVYAVTLTKSQLIHYMPI GTCATCTAAACTATTACCGCATCACTTACand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQGGCGAAACAGGAGGCAATAGCCCTGTCC terminal sequence ID NO: 91)AGGAGTTCACTGTGCCTGGTCGTGGTGTG with His6 tag)ACAGCTACCATCAGCGGCCTTAAACCTGG CGTTGATTATACCATCACTGTGTATGCTGTCACTCTGACTAAGTCTCAGCTGATACATT ACATGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA CCACCACCAC (SEQ ID NO: 135) 2525_F07MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 13WSLPYAAHMNYYRITYGETGGN GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATCAGCTGGTCTCTGCCGTATGCT AdNT1 (underlined)VDYTITVYAVTLTRSQVIHYMPI GCTCACATGAACTATTACCGCATCACTTAand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQCGGCGAAACAGGAGGCAATAGCCCTGTC terminal sequence ID NO: 92)CAGGAGTTCACTGTGCCTGGTCGTGGTGT with His6 tag)TACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCACTTTGACTAGATCACAGGTGATTCA TTACATGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATC ACCACCACCAC (SEQ ID NO: 136) 2987_A06MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 14WSLPHAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTLTKSKIIHYMPIS GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 93)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCAAAAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT(SEQ ID NO: 137) 2987_B04MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 15WSLPYPGHLNYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATCCG AdNT1 (underlined)VDYTITVYAVTLTKSKIIHYMPIS GGTCATCTGAATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHH (SEQ GGTGAAACCGGTGGTAATAGTCCGGTTCAterminal sequence ID NO: 94) GGAATTCACCGTTCCGGGTCGTGGTGTTAwith His6 tag) CCGCAACCATTAGCGGTCTGAAACCGGGTGTTGATTACACCATTACCGTTTATGCAGTT ACCCTGACCAAAAGCAAAATTATTCATTATATGCCGATTAGCATTAATTATCGCACCG AAATTGATAAACCGAGCCAGCATCATCATCACCATCAT(SEQ ID NO: 138) 2987_B09 MGVSDVPRDLEVVAATPTSLLISATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 16 WTLPHAGRAHYYRITYGETGGNGGAAGTTGTTGCAGCAACCCCGACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATTAGCTGGACCCTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTLTRSKIIHYMPIS GGTCGTGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 95)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCCGCAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 139) 2987_C02MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 17WSLPYAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATGCA AdNT1 (underlined)VDYTITVYAVTLTKSKIIHYMPIS GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 96)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCAAAAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 140) 2987_D05MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 18WSLPHAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTLTRSKIIHYMPIS GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 97)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCCGCAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 141) 2987_E03MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 19WSLPYPGHLNYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATCCG AdNT1 (underlined)VDYTITVYAVTLTRSKIIHYMPIS GGTCATCTGAATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 98)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCCGCAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 142) 2987_E08MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 20WTLPHAGRAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGACCCTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTVSSTKVIHYKPIS GGTCGTGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 99)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCGTTAGCAGCACCAAAGTGATTCATTA TAAACCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 143) 2987_F01MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 21WSLPYAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATGCA AdNT1 (underlined)VDYTITVYAVTLTRSKIIHYMPIS GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 100)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCCGCAGCAAAATTATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 144) 2987_F06MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 22WSLPHAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTLTKSKVIHYMPI GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 101)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCAAAAGCAAAGTGATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 145) 2987_G04MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 23WSLPYPGHLNYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATCCG AdNT1 (underlined)VDYTITVYAVTLTKSKVIHYMPI GGTCATCTGAATTATTATCGTATTACCTATand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 102)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCAAAAGCAAAGTGATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 146) 2987_G09MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 24WTLPHAGRAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGACCCTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTVSSTKVIVYLPIS GGTCGTGCACATTATTATCGTATTACCTATand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 103)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCGTTAGCAGCACCAAAGTTATTGTTTA TCTGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 147) 2987_H02MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 25WSLPYAGHAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGTCACTGCCGTATGCA AdNT1 (underlined)VDYTITVYAVTLTKSKVIHYMPI GGTCATGCACATTATTATCGTATTACCTATand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 104)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCCTGACCAAAAGCAAAGTGATTCATTA TATGCCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 148) 2987_H07MGVSDVPRDLEVVAATPTSLLIS ATGGGTGTTAGTGATGTTCCGCGTGATCT (Adnectin core 26WTLPHAGRAHYYRITYGETGGN GGAAGTTGTTGCAGCAACCCCGACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATTAGCTGGACCCTGCCGCATGCA AdNT1 (underlined)VDYTITVYAVTVTTTKVIHYKPI GGTCGTGCACATTATTATCGTATTACCTATand AdCT1 (italics) SINYRTEIDKPSQHHHHHH (SEQGGTGAAACCGGTGGTAATAGTCCGGTTCA terminal sequence ID NO: 105)GGAATTCACCGTTCCGGGTCGTGGTGTTA with His6 tag)CCGCAACCATTAGCGGTCTGAAACCGGGT GTTGATTACACCATTACCGTTTATGCAGTTACCGTTACCACCACCAAAGTGATTCATTA TAAACCGATTAGCATTAATTATCGCACCGAAATTGATAAACCGAGCCAGCATCATCAT CACCATCAT (SEQ ID NO: 149) 3006_A10MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 27WDAPGGLARYYRITYGETGGNS GGAAGTGGTTGCTGCCACCCCCACCAGCC AdNT1 (underlined)PVQEFTVIGRGSTATISGLKPGVD TGCTGATCAGCTGGGACGCTCCGGGTGGT sequence havingYTITVYAVTIDRDGVNHFAPISIN CTGGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) YRTEIDKPSQHHHHHH (SEQ IDCGAAACAGGAGGCAATAGCCCTGTCCAG terminal sequence NO: 106GAGTTCACTGTGATCGGTCGTGGTAGCAC with His6 tag)AGCTACCATCAGCGGCCTTAAACCTGGCG TTGATTATACCATCACTGTGTATGCTGTCACTATCGACCGTGACGGTGTCAACCACTTC GCCCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACC ACCACCAC (SEQ ID NO: 150) 3007_B08MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 28WSLPTPGLAHYYRITYGETGGNS GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingPVQEFTVPGRGVTATISGLKPGV TGCTGATCAGCTGGTCTCTGCCGACTCCA AdNT1 (underlined)DYTITVYAVTVTHHGVIGYKPISI GGTCTCGCCCATTATTACCGCATCACTTACand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence ID NO: 107) AGGAGTTCACTGTGCCTGGTCGTGGTGTTwith His6 tag) ACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGT CACTGTCACTCATCACGGCGTCATCGGCTACAAACCAATTTCCATTAATTACCGCACA GAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 151) 3007_C09 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 29 WSLPHRGVANYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCACCGT AdNT1 (underlined)VDYTITVYAVTLTGANVIIYKPIS GGTGTCGCCAATTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 108) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTCTCACTGGAGCGAACGTCATCATCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 152) 3007_C10 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 30 WSLPSSGVAHYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVPGRGVTATISGLKPGVTGCTGATCAGCTGGTCTCTGCCGAGCAGC AdNT1 (underlined)DYTITVYAVTVTNTGVIIYKPISI GGTGTCGCCCATTATTACCGCATCACTTAand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 109) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTCACTAACACTGGTGTCATCATCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 153) 3008_A03 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 31 WSLPHHGFGHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCATCAC AdNT1 (underlined)VDYTITVYAVTVTATGIIIYKPISI GGTTTCGGCCATTATTACCGCATCACTTACand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence ID NO: 110) AGGAGTTCACTGTGCCTGGTCGTGGTGTTwith His6 tag) ACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGT CACTGTCACTGCTACGGGGATCATCATCTACAAACCAATTTCCATTAATTACCGCACA GAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 154) 3008_B08 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 32 WSLPHAGDAHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCACGCC AdNT1 (underlined) VDYTITVYAVTVTRAGFYRYKPIGGTGACGCCCATTATTACCGCATCACTTA and AdCT1 (italics)SINYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTC terminal sequenceID NO: 111) CAGGAGTTCACTGTGCCTGGTCGTGGTGT with His6 tag)TACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCACTGTTACTAGAGCGGGTTTCTACCG CTACAAACCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCAC (SEQ ID NO: 155) 3008_D04MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 33WSLPHNGVAHYYRITYGETGGN GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingSPVQEFTVPGRGVTATISGLKPG TGCTGATCAGCTGGTCTCTGCCGCATAAT AdNT1 (underlined)VDYTITVYAVTVTREEVISYKPIS GGTGTCGCCCATTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 112) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTCACTCGGGAGGAAGTCATCAGCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC(SEQ ID NO: 156) 3008_F01 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 34 WSLPRQGLANYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCGTCAG AdNT1 (underlined)VDYTITVYAVTVTAAGVIIYKPIS GGTCTCGCCAATTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 113) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTCACTGCTGCTGGGGTCATCATCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 157) 3008_G01 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 35 WSLPGPGHFHYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVPGRGVTATISGLKPGVTGCTGATCAGCTGGTCTCTGCCGGGACCG AdNT1 (underlined)DYTITVYAVTVTANQPIIYKPISI GGTCACTTCCATTATTACCGCATCACTTACand AdCT1 (italics) NYRTEIDKPSQQHHHHHH (SEQ GGCGAAACAGGAGGCAATAGCCCTGTCCterminal sequence ID NO: 114) AGGAGTTCACTGTGCCTGGTCGTGGTGTTwith His6 tag) ACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGT CACTGTCACTGCTAACCAGCCCATCATCTACAAACCAATTTCCATTAATTACCGCACA GAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 158) 3008_G33 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 36 WSLPHPGLGHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCACCCC AdNT1 (underlined)VDYTITVYAVTITPETIIVYKPISI GGTCTCGGCCATTATTACCGCATCACTTAand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 115) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTATCACTCCGGAAACGATCATCGTCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 159) 3115_D04 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 37 WDAPRGLARYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVFGRGTTATISGLKPGVTGCTGATCAGCTGGGACGCTCCGAGAGGT AdNT1 (underlined)DYTITVYAVTIDRDGTRSFDPISI CTGGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 116) GAGTTCACTGTGTTCGGTCGTGGTACCACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTATCGACCGTGACGGTACCCGCAGCTTCGACCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 160) 3115_E06 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 38 WDAPAGLARYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVVGRGNTATISGLKPGVTGCTGATCAGCTGGGACGCTCCGGCTGGT AdNT1 (underlined)DYTITVYAVTIFRDGPVTWDPISI CTGGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 117) GAGTTCACTGTGGTCGGTCGTGGTAACACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTGACGGTCCCGTCACCTGGGACCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 161) 3116_A06 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 39 WSLPHQGKANYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCACCAA AdNT1 (underlined) VDYTITVYAVTVTDTGYLKYKPIGGTAAAGCCAATTATTACCGCATCACTTA and AdCT1 (italics)SINYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTC terminal sequenceID NO: 118) CAGGAGTTCACTGTGCCTGGTCGTGGTGT with His6 tag)TACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCACTGTTACTGATACAGGGTACCTCAA GTACAAACCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCAC (SEQ ID NO: 162) 3116_A07MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 40WDAPKGLARYYRITYGETGGNS GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingPVQEFTVVGRGNTATISGLKPGV TGCTGATCAGCTGGGACGCTCCGAAGGGT AdNT1 (underlined)DYTITVYAVTIFRDGPVTWDPISI CTGGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 119) GAGTTCACTGTGGTCGGTCGTGGTAACACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTGACGGTCCCGTCACCTGGGACCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 163) 3116_C01 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 41 WSLPNPGIAHYYRITYGETGGNSGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having PVQEFTVPGRGVTATISGLKPGVTGCTGATCAGCTGGTCTCTGCCGAATCCC AdNT1 (underlined)DYTITVYAVTLTGSDTIFYKPISI GGTATCGCCCATTATTACCGCATCACTTAand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 120) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTCTCACTGGCAGTGACACCATCTTCTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 164) 3116_C06 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 42 WSLPRPGNAHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGCGGCCG AdNT1 (underlined) VDYTITVYAVTVTGKDVIKYKPIGGTAACGCCCATTATTACCGCATCACTTA and AdCT1 (italics)SINYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTC terminal sequenceID NO: 121) CAGGAGTTCACTGTGCCTGGTCGTGGTGT with His6 tag)TACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCACTGTTACTGGCAAAGATGTCATCAA GTACAAACCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCAC (SEQ ID NO: 165) 3116_H06MGVSDVPRDLEVVAATPTSLLIS ATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 43WDAPAGLARYYRITYGETGGNS GGAAGTGGTTGCTGCCACCCCCACCAGCC sequence havingPVQEFTVVGRGNTATISGLKPGV TGCTGATCAGCTGGGACGCTCCGGCTGGT AdNT1 (underlined)DYTITVYAVTIFRDGVVNYGPISI CTGGCTCGATATTACCGCATCACTTACGGand AdCT1 (italics) NYRTEIDKPSQHHHHHH (SEQ CGAAACAGGAGGCAATAGCCCTGTCCAGterminal sequence ID NO: 122) GAGTTCACTGTGGTCGGTCGTGGTAACACwith His6 tag) AGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTGACGGTGTCGTCAACTACGGCCCAATTTCCATTAATTACCGCACAGA AATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 166) 3146_A08 MGVSDVPRDLEVVAATPTSLLISATGGGAGTTTCTGATGTGCCGCGCGACCT (Adnectin core 44 WSLPNPGNAHYYRITYGETGGNGGAAGTGGTTGCTGCCACCCCCACCAGCC sequence having SPVQEFTVPGRGVTATISGLKPGTGCTGATCAGCTGGTCTCTGCCGAATCCG AdNT1 (underlined)VDYTITVYAVTVTDTGFITYKPIS GGTAACGCCCATTATTACCGCATCACTTAand AdCT1 (italics) INYRTEIDKPSQHHHHHH (SEQ CGGCGAAACAGGAGGCAATAGCCCTGTCterminal sequence ID NO: 123) CAGGAGTTCACTGTGCCTGGTCGTGGTGTwith His6 tag) TACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCACTGTTACTGACACAGGTTTCATCACGTACAAACCAATTTCCATTAATTACCGCA CAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 167)Cross-Competing Adnectins and/or Adnectins that Bind to the SameAdnectin Binding Site

In one embodiment, Adnectins of the invention compete (e.g.,cross-compete) for binding to myostatin with the particularanti-myostatin Adnectins described herein. Such competing Adnectins canbe identified based on their ability to competitively inhibit binding tomyostatin of Adnectins described herein in standard myostatin bindingassays. For example, standard ELISA assays can be used in which arecombinant myostatin protein is immobilized on the plate, one of theAdnectins is fluorescently labeled and the ability of non-labeledAdnectins to compete off the binding of the labeled Adnectin isevaluated.

In one embodiment, a competitive ELISA format can be performed todetermine whether two anti-myostatin Adnectins bind overlapping epitopeson myostatin. In one format, Adnectin #1 is coated on a plate, which isthen blocked and washed. To this plate is added either myostatin alone,or myostatin pre-incubated with a saturating concentration of Adnectin#2. After a suitable incubation period, the plate is washed and probedwith a polyclonal anti-myostatin antibody, such as a biotinylated goatanti-myostatin polyclonal antibody (R&D Systems), followed by detectionwith streptavidin-HRP conjugate and standard tetramethylbenzidinedevelopment procedures. If the OD signal is the same with or withoutpreincubation with Adnectin #2, then the two Adnectins bindindependently of one another, and their epitopes do not overlap. If,however, the OD signal for wells that received myostatin/Adnectin#2mixtures is lower than for those that received myostatin alone, thenbinding of Adnectin #2 is confirmed to block binding of Adnectin #1 tomyostatin.

Alternatively, a similar experiment is conducted by surface plasmonresonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on an SPRchip surface, followed by injections of either myostatin alone ormyostatin pre-incubated with a saturating concentration of Adnectin #2.If the binding signal for myostatin/Adnectin#2 mixtures is the same orhigher than that of myostatin alone, then the two Adnectins bindindependently of one another, and their epitopes do not overlap. If,however, the binding signal for myostatin/Adnectin#2 mixtures is lowerthan the binding signal for myostatin alone, then binding of Adnectin #2is confirmed to block binding of Adnectin #1 to myostatin. A feature ofthese experiments is the use of saturating concentrations of Adnectin#2. If myostatin is not saturated with Adnectin #2, then the conclusionsabove do not hold. Similar experiments can be used to determine if anytwo myostatin binding proteins bind to overlapping epitopes.

Both assays exemplified above may also be performed in the reverse orderwhere Adnectin#2 is immobilized and myostatin—Adnectin#1 are added tothe plate. Alternatively, Adnectin #1 and/or #2 can be replaced with amonoclonal antibody and/or soluble receptor-Fc fusion protein.

In another embodiment, competition can be determined using a HTRFsandwich assay, as described in Example 4.

In other embodiments, the competing Adnectin is an Adnectin that bindsto the same Adnectin binding site on myostatin as a particularanti-myostatin Adnectin described herein. Standard mapping techniques,such as protease mapping, mutational analysis, x-ray crystallography and2-dimensional nuclear magnetic resonance, can be used to determinewhether an Adnectin binds to the same Adnectin binding site as areference Adnectin (see, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

Candidate competing anti-myostatin Adnectins can inhibit the binding ofanti-myostatin Adnectins of the invention to myostatin by at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99%. The % competition can be determined usingthe methods described above.

In some embodiments, molecules that compete with the anti-myostatinAdnectins of the invention need not be an Adnectin, but can be any typeof molecule that binds to myostatin, such as, but not limited to, anantibody, a small molecule, a peptide, and the like.

II. Extension Sequences

In certain embodiments, the anti-myostatin Adnectin molecules of thepresent invention may be modified to comprise an N-terminal extensionsequence and/or a C-terminal extension. For example, an MG sequence maybe placed at the N-terminus of the ¹⁰Fn3 defined by SEQ ID NO: 4. The Mwill usually be cleaved off, leaving a G at the N-terminus.Alternatively, the first 10 amino acids of the anti-myostatin Adnectinsshown in Table 2 may be replaced with an alternative N-terminalsequence, referred to herein as N-terminal extensions, as shown in Table7. In addition, an M, G or MG may also be placed N-terminal to any ofthe N-terminal extensions shown in Table 7. The anti-myostatin Adnectinsdescribed herein may also comprise alternative C-terminal tailsequences, referred to herein as C-terminal extension sequences. Forexample, the anti-myostatin Adnectin sequences shown in Table 2 may betruncated at the threonine corresponding to T94 of SEQ ID NO: 4 (i.e.,truncated after INYRT (SEQ ID NO: 168) portion of the sequence). Suchtruncated version may be used as therapeutic molecules in the truncatedform, or alternative C-terminal extensions may be added after thethreonine residue. Exemplary C-terminal extension sequences are shown inTable 7. Exemplary anti-myostatin Adnectins comprising C-terminalextension sequences are shown in Table 2 as SEQ ID NOs: 80-123. Forexample, SEQ ID NO: 80 (clone 1979_B06) comprises the naturallyoccurring C-terminal extension EIDKPSQ (SEQ ID NO: 211) followed by aHis6 tag (SEQ ID NO: 328). However, it should be understood that theHis6 tag is completely optional.

In certain embodiments, the C-terminal extension sequences (also called“tails”), comprise E and D residues, and may be between 8 and 50, 10 and30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In someembodiments, tail sequences include ED-based linkers in which thesequence comprises tandem repeats of ED. In exemplary embodiments, thetail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 EDrepeats. In certain embodiments, the ED-based tail sequences may alsoinclude additional amino acid residues, such as, for example: EI, EID,ES, EC, EGS, and EGC. Such sequences are based, in part, on knownAdnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 211), in whichresidues D and K have been removed. In exemplary embodiments, theED-based tail comprises an E, I or EI residues before the ED repeats.

In other embodiments, the N- or C-terminal sequences may be combinedwith known linker sequences (e.g., SEQ ID NO: 181-227 in Table 4) asnecessary when designing an anti-myostatin Adnectin fusion molecule. Insome embodiments, sequences may be placed at the C-terminus of the ¹⁰Fn3domain to facilitate attachment of a pharmacokinetic moiety. Forexample, a cysteine containing linker such as GSGC (SEQ ID NO: 189) maybe added to the C-terminus to facilitate site directed PEGylation on thecysteine residue. Exemplary anti-myostatin Adnectins comprising acysteine containing linker are shown in Table 5 as SEQ ID NOs: 228-239.

III. Pharmacokinetic Moieties

In one aspect, the application provides for anti-myostatin Adnectinsfurther comprising a pharmacokinetic (PK) moiety. Improvedpharmacokinetics may be assessed according to the perceived therapeuticneed. Often it is desirable to increase bioavailability and/or increasethe time between doses, possibly by increasing the time that a proteinremains available in the serum after dosing. In some instances, it isdesirable to improve the continuity of the serum concentration of theprotein over time (e.g., decrease the difference in serum concentrationof the protein shortly after administration and shortly before the nextadministration). The anti-myostatin Adnectin may be attached to a moietythat reduces the clearance rate of the polypeptide in a mammal (e.g.,mouse, rat, or human) by greater than two-fold, greater than three-fold,greater than four-fold or greater than five-fold relative to theunmodified anti-myostatin Adnectin. Other measures of improvedpharmacokinetics may include serum half-life, which is often dividedinto an alpha phase and a beta phase. Either or both phases may beimproved significantly by addition of an appropriate moiety. Forexample, the PK moiety may increase the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

Moieties that slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties (e.g.,polyethylene glycol), sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc and fragments and variants thereof,transferrin, or serum albumin). The anti-myostatin Adnectin may also befused to albumin or a fragment (portion) or variant of albumin asdescribed in U.S. Publication No. 2007/0048282, or may be fused to oneor more serum albumin binding Adnectin, as described herein.

Other PK moieties that can be used in the invention include thosedescribed in Kontermann et al., (Current Opinion in Biotechnology 2011;22:868-76), herein incorporated by reference. Such PK moieties include,but are not limited to, human serum albumin fusions, human serum albuminconjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb,ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based onthe three amino acids proline, alanine, and serine), carbohydrateconjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialicacid conjugates, and fatty acid conjugates.

Accordingly, in some embodiments the invention provides ananti-myostatin Adnectin fused to a PK moiety that is a polymeric sugar.In some embodiments, the PK moiety is a polyethylene glycol moiety or anFc region. In some embodiments the PK moiety is a serum albumin bindingprotein such as those described in U.S. Publication Nos. 2007/0178082and 2007/0269422. In some embodiments the PK moiety is human serumalbumin. In some embodiments, the PK moiety is transferrin.

Polyethylene Glycol

In some embodiments, the anti-myostatin Adnectin comprises polyethyleneglycol (PEG). PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161). The term “PEG” is used broadly to encompass any polyethyleneglycol molecule, without regard to size or to modification at an end ofthe PEG, and can be represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH, where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. PEG can contain furtherchemical groups which are necessary for binding reactions, which resultfrom the chemical synthesis of the molecule; or which act as a spacerfor optimal distance of parts of the molecule. In addition, such a PEGcan consist of one or more PEG side-chains which are linked together.PEGs with more than one PEG chain are called multiarmed or branchedPEGs. Branched PEGs are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462.

One or more PEG molecules may be attached at different positions on theprotein, and such attachment may be achieved by reaction with amines,thiols or other suitable reactive groups. The amine moiety may be, forexample, a primary amine found at the N-terminus of a polypeptide or anamine group present in an amino acid, such as lysine or arginine. Insome embodiments, the PEG moiety is attached at a position on thepolypeptide selected from the group consisting of: a) the N-terminus; b)between the N-terminus and the most N-terminal beta strand or beta-likestrand; c) a loop positioned on a face of the polypeptide opposite thetarget-binding site; d) between the C-terminus and the most C-terminalbeta strand or beta-like strand; and e) at the C-terminus.

PEGylation may be achieved by site-directed PEGylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere PEGylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site-directed PEGylation on the cysteine. Mutations may beintroduced into a protein coding sequence to generate cysteine residues.This might be achieved, for example, by mutating one or more amino acidresidues to cysteine. Preferred amino acids for mutating to a cysteineresidue include serine, threonine, alanine and other hydrophilicresidues. Preferably, the residue to be mutated to cysteine is asurface-exposed residue. Algorithms are well-known in the art forpredicting surface accessibility of residues based on primary sequenceor a protein. Alternatively, surface residues may be predicted bycomparing the amino acid sequences of binding polypeptides, given thatthe crystal structure of the framework, based on which bindingpolypeptides are designed and evolved, has been solved (see Himanen etal., Nature 2001; 414:933-8) and thus the surface-exposed residuesidentified. PEGylation of cysteine residues may be carried out using,for example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide.

The PEG is typically activated with a suitable activating groupappropriate for coupling to a desired site on the polypeptide.PEGylation methods are well-known in the art and further described inZalipsky, S., et al., “Use of Functionalized Poly(Ethylene Glycols) forModification of Polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press,New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16:157-182.

PEG may vary widely in molecular weight and may be branched or linear.Typically, the weight-average molecular weight of PEG is from about 100Daltons to about 150,000 Daltons. Exemplary weight-average molecularweights for PEG include about 20,000 Daltons, about 40,000 Daltons,about 60,000 Daltons and about 80,000 Daltons. In certain embodiments,the molecular weight of PEG is 40,000 Daltons. Branched versions of PEGhaving a total molecular weight of any of the foregoing can also beused. In some embodiments, the PEG has two branches. In otherembodiments, the PEG has four branches. In another embodiment, the PEGis a bis-PEG (NOF Corporation, DE-200MA), in which two Adnectins areconjugated (see, e.g., Example 1 and ATI-1341 of Table 5).

Conventional separation and purification techniques known in the art canbe used to purify PEGylated anti-myostatin Adnectins, such as sizeexclusion (e.g., gel filtration) and ion exchange chromatography.Products may also be separated using SDS-PAGE. Products that may beseparated include mono-, di-, tri-, poly- and un-PEGylated Adnectins, aswell as free PEG. The percentage of mono-PEG conjugates can becontrolled by pooling broader fractions around the elution peak toincrease the percentage of mono-PEG in the composition. About 90%mono-PEG conjugates represent a good balance of yield and activity.

In some embodiments, the PEGylated anti-myostatin Adnectins willpreferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%or 100% of the biological activity associated with the unmodifiedanti-myostatin Adnectin. In some embodiments, biological activity refersto its ability to bind to myostatin, as assessed by K_(D), k_(on), ork_(off). In some embodiments, the PEGylated anti-myostatin Adnectinshows an increase in binding to myostatin relative to unPEGylatedanti-myostatin Adnectin.

Exemplary PEG-modified anti-myostatin Adnectins are shown in Table 5.

Immunoglobulin Fc Domain (and Fragments)

In some embodiments, the anti-myostatin Adnectin is fused to animmunoglobulin Fc domain, or a fragment or variant thereof. As usedherein, a “functional Fc region” is an Fc domain or fragment thereofwhich retains the ability to bind FcRn. In some embodiments, afunctional Fc region binds to FcRn, bud does not possess effectorfunction. The ability of the Fc region or fragment thereof to bind toFcRn can be determined by standard binding assays known in the art. Inother embodiments, the Fc region or fragment thereof binds to FcRn andpossesses at least one “effector function” of a native Fc region.Exemplary “effector functions” include C1q binding; complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an anti-myostatin Adnectin) and can be assessed usingvarious assays known in the art for evaluating such antibody effectorfunctions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification. Preferably, the variant Fc region has at least one aminoacid substitution compared to a native sequence Fc region or to the Fcregion of a parent polypeptide, e.g., from about one to about ten aminoacid substitutions, and preferably from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. The variant Fc region herein will preferablypossess at least about 80% sequence identity with a native sequence Fcregion and/or with an Fc region of a parent polypeptide, and mostpreferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

In an exemplary embodiment, the Fc domain is derived from an IgG1subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) mayalso be used. Shown below is the sequence of a human IgG1 immunoglobulinFc domain:

(SEQ ID NO: 169) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

The core hinge sequence is underlined, and the CH2 and CH3 regions arein regular text. It should be understood that the C-terminal lysine isoptional.

The fusion may be formed by attaching an anti-myostatin Adnectin toeither end of the Fc molecule, i.e., Fc-anti-myostatin Adnectin oranti-myostatin Adnectin-Fc arrangements. In certain embodiments, the Fcand anti-myostatin Adnectin are fused via a linker. Exemplary linkersequences include

(SEQ ID NO: 181) GAGGGGSG, (SEQ ID NO: 182) EPKSSD, D, (SEQ ID NO: 183)ESPKAQASSVPTAQPQAEGLA, (SEQ ID NO: 184) ELQLEESAAEAQDGELD,(SEQ ID NO: 185) GQPDEPGGS, (SEQ ID NO: 186) GGSGSGSGSGSGS,(SEQ ID NO: 187) ELQLEESAAEAQEGELE, (SEQ ID NO: 188) GSGSG,(SEQ ID NO: 189) GSGC, (SEQ ID NO: 190) AGGGGSG, (SEQ ID NO: 191) GSGS,(SEQ ID NO: 192) QPDEPGGS, (SEQ ID NO: 193) GSGSGS, (SEQ ID NO: 194)TVAAPS, (SEQ ID NO: 195) KAGGGGSG, (SEQ ID NO: 196) KGSGSGSGSGSGS,(SEQ ID NO: 197) KQPDEPGGS, (SEQ ID NO: 198) KELQLEESAAEAQDGELD,(SEQ ID NO: 199) KTVAAPS, (SEQ ID NO: 200) KAGGGGSGG, (SEQ ID NO: 201)KGSGSGSGSGSGSG, (SEQ ID NO: 202) KQPDEPGGSG, (SEQ ID NO: 203)KELQLEESAAEAQDGELDG, (SEQ ID NO: 204) KTVAAPSG (SEQ ID NO: 205)AGGGGSGG, (SEQ ID NO: 206) AGGGGSG, (SEQ ID NO: 207) GSGSGSGSGSGSG,(SEQ ID NO: 208) QPDEPGGSG, and (SEQ ID NO: 209) TVAAPSG.

In some embodiments, the Fc region used in the anti-myostatin Adnectinfusion comprises the hinge region of an Fc molecule. As used herein, the“hinge” region comprises the core hinge residues spanning positions 1-16of SEQ ID NO: 169 (DKTHTCPPCPAPELLG; SEQ ID NO: 170) of the IgG1 Fcregion. In certain embodiments, the anti-myostatin Adnectin-Fc fusionadopts a multimeric structure (e.g., dimer) owing, in part, to thecysteine residues at positions 6 and 9 of SEQ ID NO: 169 within thehinge region. In other embodiments, the hinge region as used herein, mayfurther include residues derived from the CH1 and CH2 regions that flankthe core hinge sequence, as shown in SEQ ID NO: 169. In yet otherembodiments, the hinge sequence is GSTHTCPPCPAPELLG (i.e., hingesequence for PRD-932; SEQ ID NO: 180).

In some embodiments, the hinge sequence, may include substitutions thatconfer desirable pharmacokinetic, biophysical, and/or biologicalproperties. Some exemplary hinge sequences include

EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 171;core hinge region underlined),

EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO 172;core hinge region underlined),

EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 173;core hinge region underlined),

DKTHTCPPCPAPELLGGPS (SEQ ID NO: 174;core hinge region underlined), and

DKTHTCPPCPAPELLGGSS (SEQ ID NO: 175;core hinge region underlined). In one embodiment, the residue P atposition 18 of SEQ ID NO: 169 has been replaced with S to ablate Fceffector function; this replacement is exemplified in hinges having anyone of SEQ ID NOs: 172, 173, and 175. In another embodiment, theresidues DK at positions 1-2 of SEQ ID NO: 169 have been replaced withGS to remove a potential clip site; this replacement is exemplified inSEQ ID NO: 173. In another embodiment, the C at position 103 of SEQ IDNO: 176, which corresponds to the heavy chain constant region of humanIgG1 (i.e., domains CH1-CH3), has been replaced with S to preventimproper cysteine bond formation in the absence of a light chain; thisreplacement is exemplified in SEQ ID NOs: 171-173.

(SEQ ID NO: 176) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

In certain embodiments, an anti-myostatin Adnectin-Fc fusion may havethe following configurations: 1) anti-myostatin Adnectin-hinge-Fc or 2)hinge-Fc-anti-myostatin Adnectin. Therefore, any anti-myostatin Adnectinof the present invention can be fused to an Fc region comprising a hingesequence according to these configurations. In some embodiments, alinker may be used to join the anti-myostatin Adnectin to the hinge-Fcmoiety, for example, an exemplary fusion protein may have theconfiguration anti-myostatin Adnectin-linker-hinge-Fc orhinge-Fc-linker-anti-myostatin Adnectin. Additionally, depending on thesystem in which the fusion polypeptide is produced, a leader sequencemay placed at the N-terminus of the fusion polypeptide. For example, ifthe fusion is produced in a mammalian system, a leader sequence such asMETDTLLLWVLLLWVPGSTG (SEQ ID NO: 177) may be added to the N-terminus ofthe fusion molecule. If the fusion is produced in E. coli, the fusionsequence will be preceded by a methionine.

The following sequence exemplifies an anti-myostatin Adnectin-hinge-Fcconstruct:

(PRD-1171; SEQ ID NO: 253) GVSDVPRDLEVVAATPTSLLISWTLPHAGRAHYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTTTKVIHYKPISINYRTEIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK.The leader sequence is in bold, the anti-myostatin Adnectin sequence isin italics, and the hinge region is underlined. It should be understoodthat the C-terminal lysine is optional.

Here, the Fc domain comprises the human IgG1 CH2 and CH3 regions asfollows:

(SEQ ID NO: 178) VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKand the hinge sequence

(SEQ ID NO: 170) DKTHTCPPCPAPELLG.

The following sequence exemplifies an Fc-anti-myostatin Adnectinconstruct:

(PRD-1474; SEQ ID NO: 273) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP ELQLEESAAEAQEGELEGVSDVPRDLEVVAATPTSLLISWSLPHQ GKANYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTDTGYLKYKPISINYRTEI.The hinge region is underlined, the leader sequence is in bold, and theanti-myostatin Adnectin sequence is in italics.

Here, the Fc domain comprises the human IgG1 CH2 and CH3 regions asfollows:

(SEQ ID NO: 179) VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPand the hinge sequence

(SEQ ID NO: 170) DKTHTCPPCPAPELLG.

Exemplary anti-myostatin Adnectin-Fc fusions and Fc-anti-myostatinAdnectin fusions are shown in Table 6 (SEQ ID NOs: 252-273). Allsequences may begin with a methionine or a mammalian leader sequence(e.g., SEQ ID NO: 177).

Adnectins

In some embodiments the PK moiety is another Adnectin specific, forexample, to a serum protein (e.g., human serum albumin), as described inUS 2012/0094909, herein incorporated by reference in its entirety. OtherPK moieties that may be used with the Adnectins of the invention aredisclosed in Kontermann et al. (Current Opinion in Biotechnology 2011;22:868-76), as discussed supra. By way of example, such Adnectin basedPK moieties may be directly or indirectly linked to an anti-myostatinAdnectin via a polypeptide linker. Suitable linkers for joining Fn3domains are those which allow the separate domains to fold independentlyof each other and form a three dimensional structure that permits highaffinity binding to a target molecule. Exemplary polypeptide linkersinclude PSTSTST (SEQ ID NO: 210), EIDKPSQ (SEQ ID NO: 211), and GSlinkers, such as GSGSGSGSGS (SEQ ID NO: 213) and multimers thereof. Insome embodiments, the linker is a glycine-serine based linker. Theselinkers comprise glycine and serine residues and may be between 8 and50, 10 and 30, and 10 and 20 amino acids in length. Examples includelinkers having an amino acid sequence (GS)₇ (SEQ ID NO: 215), G(GS)₆(SEQ ID NO: 216), and G(GS)₇G (SEQ ID NO: 217). Other linkers containglutamic acid, and include, for example, (GSE)₅ (SEQ ID NO: 218) andGGSEGGSE (SEQ ID NO: 219). Other exemplary glycine-serine linkersinclude (GS)₄ (SEQ ID NO: 212), (GGGGS)₇ (SEQ ID NO: 220), (GGGGS)₅ (SEQID NO: 221), and (GGGGS)₃G (SEQ ID NO: 222). In some embodiments, thelinker is a glycine-proline based linker. These linkers comprise glycineand proline residues and may be between 3 and 30, 10 and 30, and 3 and20 amino acids in length. Examples include linkers having an amino acidsequence (GP)₃G (SEQ ID NO: 223), (GP)₅G (SEQ ID NO: 224), and GPG. Inother embodiments, the linker may be a proline-alanine based linkerhaving between 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples of proline alanine based linkers include, for example, (PA)₃(SEQ ID NO: 225), (PA)₆ (SEQ ID NO: 226) and (PA)₉ (SEQ ID NO: 227).Optimal linker length and amino acid composition may be determined byroutine experimentation in view of the teachings provided herein. Insome embodiments, an anti-myostatin Adnectin is linked, for example, toan anti-HSA Adnectin via a polypeptide linker having a protease sitethat is cleavable by a protease in the blood or target tissue. Suchembodiments can be used to release an anti-myostatin Adnectin for betterdelivery or therapeutic properties or more efficient production.

Additional linkers or spacers, may be introduced at the N-terminus orC-terminus of a Fn3 domain between the Fn3 domain and the polypeptidelinker.

In some embodiments, an anti-myostatin Adnectin may be directly orindirectly linked for example, to an anti-HSA Adnectin via a polymericlinker. Polymeric linkers can be used to optimally vary the distancebetween each component of the fusion to create a protein fusion with oneor more of the following characteristics: 1) reduced or increased sterichindrance of binding of one or more protein domains when binding to aprotein of interest, 2) increased protein stability or solubility, 3)decreased protein aggregation, and 4) increased overall avidity oraffinity of the protein.

In some embodiments, an anti-myostatin Adnectin is linked, for example,to an anti-HSA Adnectin, via a biocompatible polymer such as a polymericsugar. The polymeric sugar can include an enzymatic cleavage site thatis cleavable by an enzyme in the blood or target tissue. Suchembodiments can be used to release an anti-myostatin Adnectin for betterdelivery or therapeutic properties or more efficient production.

A summary of monoAdnectins and their corresponding PK moiety modifiedforms (e.g., PEGylated and Fc fusions) are presented in Table 3.

TABLE 3 Mono- Adnectin ^(a) Cysteine mutant [modification]^(b)X-linker-Fc^(c) Fc-linker-X^(d) 1979_B06 ATI-1107 (SEQ ID NO: 229) [40k2-br] (SEQ ID NO: 80) 2062_G02 ATI-1106 (SEQ ID NO: 228) [40k 2-br] (SEQID NO: 81) 2522_C09 (SEQ ID NO: 82) 2523_G06 (SEQ ID NO: 83) 2524_C11(SEQ ID NO: 84) 2524_D09 ATI-1275 (SEQ ID NO: 231) [NEM]; (SEQ ID NO:85) ATI-1276 (SEQ ID NO: 231) [40k 2-br] 2524_E10 (SEQ ID NO: 86)2524_H05 (SEQ ID NO: 87) 2524_H11 (SEQ ID NO: 88) 2525_B01 (SEQ ID NO:89) 2525_D02 ATI-1267 (SEQ ID NO: 230) [NEM]; (SEQ ID NO: 90) ATI-1266(SEQ ID NO: 230) [40k 2-br] 2525_D05 ATI-1277 (SEQ ID NO: 232) [NEM];PRD-932 [L1] (SEQ ID NO: 91) ATI-1278 (SEQ ID NO: 232) [40k 2-br] (SEQID NO: 252) 2525_F07 (SEQ ID NO: 92) 2987_A06 (SEQ ID NO: 93) 2987_B04(SEQ ID NO: 94) 2987_B09 (SEQ ID NO: 95) 2987_C02 (SEQ ID NO: 96)2987_D05 (SEQ ID NO: 97) 2987_E03 (SEQ ID NO: 98) 2987_E08 (SEQ ID NO:99) 2987_F01 (SEQ ID NO: 100) 2987_F06 (SEQ ID NO: 101) 2987_G04 (SEQ IDNO: 102) 2987_G09 (SEQ ID NO: 103) 2987_H02 (SEQ ID NO: 104) 2987_H07ATI-1310 (SEQ ID NO: 233) [none]; PRD-1171[L2] PRD-1175[L1] (SEQ ID NO:105) ATI-1340 (SEQ ID NO: 233) [NEM]; (SEQ ID NO: 253); (SEQ ID NO:256); ATI-1338 (SEQ ID NO: 233) [40k 2-br]; PRD-1173[L3] PRD- 1177[L5]ATI-1359 (SEQ ID NO: 233) [no His, 40k 2-br]; (SEQ ID NO: 254); (SEQ IDNO: 257); ATI-1339 (SEQ ID NO: 233) [40k 4-br]; PRD-1174[L4] PRD-1178[L6] ATI-1341 (SEQ ID NO: 233) [20k bis-PEG] (SEQ ID NO: 255) (SEQID NO: 258); PRD-1180[L7] (SEQ ID NO: 259); PRD-1471[L8] (SEQ ID NO:270) 3006_A10 (SEQ ID NO: 106) 3007_B08 (SEQ ID NO: 107) 3007_C09 (SEQID NO: 108) 3007_C10 (SEQ ID NO: 109) 3008_A03 (SEQ ID NO: 110) 3008_B08(SEQ ID NO: 111) 3008_D04 (SEQ ID NO: 112) 3008_F01 (SEQ ID NO: 113)3008_G01 (SEQ ID NO: 114) 3008_G03 (SEQ ID NO: 115) 3115_D04 ATI-1375(SEQ ID NO: 235) [40k 2-br] PRD-1301[L2] PRD-1284[L5] (SEQ ID NO: 116)(SEQ ID NO: 265) (SEQ ID NO: 260) 3115_E06 ATI-1376 (SEQ ID NO: 236)[40k 2-br] PRD-1302[L2] PRD-1285[L5] (SEQ ID NO: 117) (SEQ ID NO: 266)(SEQ ID NO: 261); PRD-1472[L8] (SEQ ID NO: 271) 3116_A06 ATI-1379 (SEQID NO: 239) [40k 2-br]; PRD-1305[L2] PRD-1288[L5] (SEQ ID NO: 118)ATI-1523 (SEQ ID NO: 239) [NEM] (SEQ ID NO: 269) (SEQ ID NO: 264);PRD-1474[L8] (SEQ ID NO: 273) 3116_A07 ATI-1377 (SEQ ID NO: 237) [40k2-br] PRD-1303[L2] PRD-1286[L5] (SEQ ID NO: 119) (SEQ ID NO: 267) (SEQID NO: 262); PRD-1473[L8] (SEQ ID NO: 272) 3116_C01 (SEQ ID NO: 120)3116_C06 (SEQ ID NO: 121) 3116_H06 (SEQ ID NO: 122) 3146_A08 ATI-1378(SEQ ID NO: 238) [40k 2-br] PRD-1304[L2] PRD-1287[L5] (SEQ ID NO: 123)(SEQ ID NO: 268) (SEQ ID NO: 263) ^(a) Unmodified monoAdnectins have acore Adnectin sequence preceded by a N-terminal extension sequence(MGVSDVPRDL; SEQ ID NO: 306) and followed by a C-terminal tail(EIDKPSQHHHHHH; SEQ ID NO: 325), as shown in Table 2. The core Adnectinsequence corresponds to the monoAdnectin sequence lacking the N-terminalextension and C-terminal tail sequences. ^(b)Adnectins with cysteinemutants have the core Adnectin sequence of the monoAdnectin in the firstcolumn, and are preceded by a N-terminal extension sequence (MGVSDVPRDL;SEQ ID NO: 306) and followed by a C-terminal tail(GSGC[Modification]HHHHHH; SEQ ID NO: 326 or EGSGC[Modification]HHHHHH;SEQ ID NO: 327), as shown in Table 5. ^(c)Adnectins with an Fc moiety onthe C-terminus have the core Adnectin sequence of the monoAdnectin inthe first column, which is preceded by a N-terminal extension sequence(GVSDVPRDL; SEQ ID NO: 307) and followed by a C-terminal tail (EI),which is followed by a linker sequence (Table 4) and the Fc regionsequence, as described in Table 6. ^(d)Adnectins with an Fc moiety onthe N-terminus have an Fc region sequence which is preceded by aN-terminal hinge sequence and followed by a linker (Table 4) and thecore Adnectin sequence of the monoAdnectin in the first column, whichitself is preceded by a N-terminal extension sequence (GVSDVPRDL; SEQ IDNO: 307) and followed by a C-terminal tail (EI), as shown in Table 6.

The SEQ ID NOs of exemplary linkers of the invention are presented inTable 4.

TABLE 4 SEQ ID NO. LINKER SEQUENCE 181 L1 GAGGGGSG 182 L2 EPKSSD --- L3D 183 L4 ESPKAQASSVPTAQPQAEGLA 184 L5 ELQLEESAAEAQDGELD 185 L6 GQPDEPGGS186 L7 GGSGSGSGSGSGS 187 L8 ELQLEESAAEAQEGELEADDITIONAL EXEMPLARY LINKERS 188 L9 GSGSG 189 L10 GSGC 190 L11 AGGGGSG191 L12 GSGS 192 L13 QPDEPGGS 193 L14 GSGSGS 194 L15 TVAAPS 195 L16KAGGGGSG 196 L17 KGSGSGSGSGSGS 197 L18 KQPDEPGGS 198 L19KELQLEESAAEAQDGELD 199 L20 KTVAAPS 200 L21 KAGGGGSGG 201 L22KGSGSGSGSGSGSG 202 L23 KQPDEPGGSG 203 L24 KELQLEESAAEAQDGELDG 204 L25KTVAAPSG 205 L26 AGGGGSGG 206 L27 AGGGGSG 207 L28 GSGSGSGSGSGSG 208 L29QPDEPGGSG 209 L30 TVAAPSG 210 L31 PSTSTST 211 L32 EIDKPSQ 212 L33GSGSGSGS 213 L34 GSGSGSGSGS 214 L35 GSGSGSGSGSGS 215 L36 GSGSGSGSGSGSGS216 L37 GGSGSGSGSGSGS 217 L38 GGSGSGSGSGSGSGSG 218 L39 GSEGSEGSEGSEGSE219 L40 GGSEGGSE 220 L41 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 221 L42GGGGSGGGGSGGGGSGGGGSGGGGS 222 L43 GGGGSGGGGSGGGGSG 223 L44 GPGPGPG 224L45 GPGPGPGPGPG --- L46 GPG 225 L47 PAPAPA 226 L48 PAPAPAPAPAPA 227 L49PAPAPAPAPAPAPAPAPA

The SEQ ID NOs of exemplary PEGylated anti-myostatin Adnectins of theinvention are presented in Table 5.

TABLE 5 PEGylated Anti-Myostatin Adnectins Sequence CloneAmino Acid Sequence Nucleic Acid Sequence ATI-1106 MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT [40K 2- LLISWVSPRGRARYYRITYGGAAGTGGTTGCTGCCACCCCCACCAGCC branch PEG] GETGGNSPVQEFTVPGRGSTGCTGATCAGCTGGGTTTCTCCGCGTGGT TATISGLKPGVDYTITVYACGTGCTCGATATTACCGCATCACTTACGG VTIYRDGMSHHDPISINYRCGAAACAGGAGGCAATAGCCCTGTCCAGG TGSGC[Modification]AGTTCACTGTGCCTGGTCGTGGTTCTACA HHHHHH GCTACCATCAGCGGCCTTAAACCTGGCGT(SEQ ID NO: 228) TGATTATACCATCACTGTGTATGCTGTCACTATCTACCGTGACGGTATGTCTCATCAT GACCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 240) ATI-1107MGVSDVPRDLEVVAATPTS ATGGGAGTTTCTGATGTGCCGCGCGACCT [40K 2-LLISWSLPHAGHVNYYRIT GGAAGTGGTTGCTGCCACCCCCACCAGCC branch PEG]YGETGGNSPVQEFTVPGRG TGCTGATCAGCTGGTCTCTGCCGCATGCT VTATISGLKPGVDYTITVYGGTCATGTGAACTATTACCGCATCACTTA AVTLTKSQMIHYMPISINYCGGCGAAACAGGAGGCAATAGCCCTGTCC RTGSGC AGGAGTTCACTGTGCCTGGTCGTGGTGTT[Modification]HHHHH ACAGCTACCATCAGCGGCCTTAAACCTGG H (SEQ ID NO: 229)CGTTGATTATACCATCACTGTGTATGCTG TCACTCTGACTAAATCTCAGATGATCCATTACATGCCAATTTCCATTAATTACCGCAC AGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 241) ATI-1266 [40K 2- MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT branch PEG] LLISWTLPHAGRAHYYRITGGAAGTGGTTGCTGCCACCCCCACCAGCC ATI-1267 [N- YGETGGNSPVQEFTVPGRGTGCTGATCAGCTGGACTTTGCCGCATGCT ethylmaleimide] VTATISGLKPGVDYTITVYGGTCGTGCGCACTATTACCGCATCACTTA AVTVTTTSVILYKPISINYCGGCGAAACAGGAGGCAATAGCCCTGTCC RTEGSGC AGGAGTTCACTGTGCCTGGGCGGGGTGTT[Modification]HHHHH ACAGCTACCATCAGCGGCCTTAAACCTGG H (SEQ ID NO: 230)CGTTGATTATACCATCACTGTGTATGCTG TCACTGTGACGACAACTTCGGTGATCCTTTACAAGCCAATTTCCATTAATTACCGCAC AGAAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 242) ATI-1275 [N- MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT ethylmaleimide] LLISWYLPYPAHMNYYRITGGAAGTGGTTGCTGCCACCCCCACCAGCC ATI-1276 [40K 2- YGETGGNSPVQEFTVPGRGTGCTGATCAGCTGGTACCTCCCGTATCCT branch PEG] LTATISGLKPGVDYTITVYGCGCATATGAACTATTACCGCATCACTTA AVTLTKSQILHHRPISINYCAGGAGTTCACTGTGCCTGGGCGGGGTCT RTEGSGC GACAGCTACCATCAGCGGCCTTAAACCTG[Modification]HHHHC GCGTTGATTATACCATCACTGTGTATGCT GGCGAAACAGGAGGCAATAGTCACTCTGACAAAATCTCAGATTCTCCA GCCCTGTCHH TCATAGGCCAATTTCCATTAATTACCGCA(SEQ ID NO: 231) CAGAAGGTAGCGGTTGCCACCATCACCAC CATCAC (SEQ ID NO: 243)ATI-1277 [N- MGVSDVPRDLEVVAATPTS ATGGGAGTTTCTGATGTGCCGCGCGACCTethylmaleimide] LLISWSLPYAGHLNYYRIT GGAAGTGGTTGCTGCCACCCCCACCAGCCATI-1278 [40K 2- YGETGGNSPVQEFTVPGRG TGCTGATCAGCTGGTCTCTTCCTTATGCTbranch PEG] VTATISGLKPGVDYTITVY GGTCATCTAAACTATTACCGCATCACTTAAVTLTKSQLIHYMPISINY CAGGAGTTCACTGTGCCTGGTCGTGGTGT RTEGSGCGACAGCTACCATCAGCGGCCTTAAACCTG [Modification]HHHGGGCGTTGATTATACCATCACTGTGTATGCT CGAAACAGGAGGCAATAGCGTCACTCTGACTAAGTCTCAGCTGATACA CCTGTCCHHH TTACATGCCAATTTCCATTAATTACCGCA(SEQ ID NO: 232) CAGAAGGTAGCGGTTGCCACCATCACCAC CATCAC (SEQ ID NO: 244)ATI-1310 [free Cys] MGVSDVPRDLEVVAATPTS ATGGGTGTTAGTGATGTTCCGCGTGATCTATI-1338 [40K 2- LLISWTLPHAGRAHYYRIT GGAAGTTGTTGCAGCAACCCCGACCAGCCbranch PEG] YGETGGNSPVQEFTVPGRG TGCTGATTAGCTGGACCCTGCCGCATGCAATI-1339 [40K 4- VTATISGLKPGVDYTITVY GGTCGTGCACATTATTATCGTATTACCTAbranch PEG] AVTVTTTKVIHYKPISINY TGGTGAAACCGGTGGTAATAGTCCGGTTCATI-1340 [N- RTEGSGC AGGAATTCACCGTTCCGGGTCGTGGTGTT ethylmaleimide][Modification]HHHHH ACCGCAACCATTAGCGGTCTGAAACCGGG ATI-1341 [20K Bis-H (SEQ ID NO: 233) TGTTGATTACACCATTACCGTTTATGCAG PEG]TTACCGTTACCACCACCAAAGTGATTCAT TATAAACCGATTTCCATTAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACC ATCAC (SEQ ID NO: 245) ATI-1359 [40K 2-MGVSDVPRDLEVVAATPTS ATGGGTGTTAGTGATGTTCCGCGTGATCT branch PEG]LLISWTLPHAGRAHYYRIT GGAAGTTGTTGCAGCAACCCCGACCAGCC YGETGGNSPVQEFTVPGRGTGCTGATTAGCTGGACCCTGCCGCATGCA VTATISGLKPGVDYTITVYGGTCGTGCACATTATTATCGTATTACCTA AVTVTTTKVIHYKPISINYTGGTGAAACCGGTGGTAATAGTCCGGTTC RTEGSGC AGGAATTCACCGTTCCGGGTCGTGGTGTT[Modification]HHHHH ACCGCAACCATTAGCGGTCTGAAACCGGG H (SEQ ID NO: 234)TGTTGATTACACCATTACCGTTTATGCAG TTACCGTTACCACCACCAAAGTGATTCATTATAAACCGATTTCCATTAATTACCGAAC AGAAGGTAGCGGTTGC (SEQ ID NO: 246)ATI-1375 [40K 2- MGVSDVPRDLEVVAATPTS ATGGGAGTTTCTGATGTGCCGCGCGACCTbranch PEG] LLISWDAPRGLARYYRITY GGAAGTGGTTGCTGCCACCCCCACCAGCCGETGGNSPVQEFTVFGRGT TGCTGATCAGCTGGGACGCTCCGAGAGGT TATISGLKPGVDYTITVYACTGGCTCGATATTACCGCATCACTTACGG VTIDRDGTRSFDPISINYRCGAAACAGGAGGCAATAGCCCTGTCCAGG TEGSGC AGTTCACTGTGTTCGGTCGTGGTACCACA[Modification]HHHHH GCTACCATCAGCGGCCTTAAACCTGGCGT H (SEQ ID NO: 235)TGATTATACCATCACTGTGTATGCTGTCA CTATCGACCGTGACGGTACCCGCAGCTTCGACCCAATTTCCATTAATTACCGCACAGA AGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 247) ATI-1376 [40K 2- MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT branch PEG] LLISWDAPAGLARYYRITYGGAAGTGGTTGCTGCCACCCCCACCAGCC GETGGNSPVQEFTVVGRGNTGCTGATCAGCTGGGACGCTCCGGCTGGT TATISGLKPGVDYTITVYACTGGCTCGATATTACCGCATCACTTACGG VTIFRDGPVTWDPISINYRCGAAACAGGAGGCAATAGCCCTGTCCAGG TEGSGC AGTTCACTGTGGTCGGTCGTGGTAACACA[Modification]HHHHH GCTACCATCAGCGGCCTTAAACCTGGCGT H (SEQ ID NO: 236)TGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTGACGGTCCCGTCACCTGGGACCCAATTTCCATTAATTACCGCACAGA AGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 248) ATI-1377 [40K 2- MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT branch PEG] LLISWDAPKGLARYYRITYGGAAGTGGTTGCTGCCACCCCCACCAGCC GETGGNSPVQEFTVVGRGNTGCTGATCAGCTGGGACGCTCCGAAGGGT TATISGLKPGVDYTITVYACTGGCTCGATATTACCGCATCACTTACGG VTIFRDGPVTWDPISINYRCGAAACAGGAGGCAATAGCCCTGTCCAGG TEGSGC AGTTCACTGTGGTCGGTCGTGGTAACACA[Modification]HHHHH GCTACCATCAGCGGCCTTAAACCTGGCGT H (SEQ ID NO: 237)TGATTATACCATCACTGTGTATGCTGTCA CTATCTTCCGTGACGGTCCCGTCACCTGGGACCCAATTTCCATTAATTACCGCACAGA AGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 249) ATI-1378 [40K 2- MGVSDVPRDLEVVAATPTSATGGGAGTTTCTGATGTGCCGCGCGACCT branch PEG] LLISWSLPNPGNAHYYRITGGAAGTGGTTGCTGCCACCCCCACCAGCC YGETGGNSPVQEFTVPGRGTGCTGATCAGCTGGTCTCTGCCGAATCCG VTATISGLKPGVDYTITVYGGTAACGCCCATTATTACCGCATCACTTA AVTVTDTGFITYKPISINYCAGGAGTTCACTGTGCCTGGTCGTGGTGT RTEGSGC TACAGCTACCATCAGCGGCCTTAAACCTG[Modification]HHHHC GCGTTGATTATACCATCACTGTGTATGCT GGCGAAACAGGAGGCAATAGTCACTGTTACTGACACAGGTTTCATCAC GCCCTGTCHH GTACAAACCAATTTCCATTAATTACCGCA(SEQ ID NO: 238) CAGAAGGTAGCGGTTGCCACCATCACCAC CATCAC (SEQ ID NO: 250)ATI-1379 [40K 2- MGVSDVPRDLEVVAATPTS ATGGGAGTTTCTGATGTGCCGCGCGACCTbranch PEG] LLISWSLPHQGKANYYRIT GGAAGTGGTTGCTGCCACCCCCACCAGCCATI-1523 [N- YGETGGNSPVQEFTVPGRG TGCTGATCAGCTGGTCTCTGCCGCACCAAethylmaleimide] VTATISGLKPGVDYTITVY GGTAAAGCCAATTATTACCGCATCACTTAAVTVTDTGYLKYKPISINY CGGCGAAACAGGAGGCAATAGCCCTGTCC RTEGSGCAGGAGTTCACTGTGCCTGGTCGTGGTGTT [Modification]HHHHHACAGCTACCATCAGCGGCCTTAAACCTGG H (SEQ ID NO: 239)CGTTGATTATACCATCACTGTGTATGCTG TCACTGTTACTGATACAGGGTACCTCAAGTACAAACCAATTTCCATTAATTACCGCAC AGAAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 251)

The SEQ ID NOs of exemplary Fc-fused anti-myostatin Adnectins of theinvention are presented in Table 6.

TABLE 6 Fc-fused Anti-Myostatin Adnectins Sequence Amino Acid N-terminalC-terminal Clone Sequence domain Linker domain Nucleic Acid SequencePRD-932 EPKSSGSTHTC EPKSSGSTHTCP GAGGGGSG GVSDVPRDLEVVAGAGCCCAAATCTAGCGGGTC PPCPAPELLGG PCPAPELLGGSS (SEQ ID ATPTSLLISWSLPGACTCACACATGCCCACCGT SSVFLFPPKPK VFLFPPKPKDTL NO: 181) YAGHLNYYRITYGGCCCAGCACCTGAACTCCTG DTLMISRTPEV MISRTPEVTCVV ETGGNSPVQEFTVGGGGGAAGCTCAGTCTTCCT TCVVVDVSHED VDVSHEDPEVKF PGRGVTATISGLKCTTCCCCCCAAAACCCAAGG PEVKFNWYVDG NWYVDGVEVHNA PGVDYTITVYAVTACACCCTCATGATCTCCCGG VEVHNAKTKPR KTKPREEQYNST LTKSQLIHYMPISACCCCTGAGGTCACATGCGT EEQYNSTYRVV YRVVSVLTVLHQ INYRTEI (SEQGGTGGTGGACGTGAGCCACG SVLTVLHQDWL DWLNGKEYKCKV ID NO: 282)AAGACCCTGAGGTCAAGTTC NGKEYKCKVSN SNKALPAPIEKT AACTGGTACGTGGACGGCGTKALPAPIEKTI ISKAKGQPREPQ GGAGGTGCATAATGCCAAGA SKAKGQPREPQ VYTLPPSRDELTCAAAGCCGCGGGAGGAGCAG VYTLPPSRDEL KNQVSLTCLVKG TACAACAGCACGTACCGTGTTKNQVSLTCLV FYPSDIAVEWES GGTCAGCGTCCTCACCGTCC KGFYPSDIAVE NGQPENNYKTTPTGCACCAGGACTGGCTGAAT WESNGQPENNY PVLDSDGSFFLY GGCAAGGAGTACAAGTGCAAKTTPPVLDSDG SKLTVDKSRWQQ GGTCTCCAACAAAGCCCTCC SFFLYSKLTVD GNVFSCSVMHEACAGCCCCCATCGAGAAAACC KSRWQQGNVFS LHNHYTQKSLSL ATCTCCAAAGCCAAAGGGCACSVMHEALHNH SP (SEQ ID GCCCCGAGAACCACAGGTGT YTQKSLSLSPG NO: 274)ACACCCTGCCCCCATCCCGG AGGGGSGGVSD GATGAGCTGACCAAGAACCA VPRDLEVVAATGGTCAGCCTGACCTGCCTGG PTSLLISWSLP TCAAAGGCTTCTATCCCAGC YAGHLNYYRITGACATCGCCGTGGAGTGGGA YGETGGNSPVQ GAGCAATGGGCAGCCGGAGA EFTVPGRGVTAACAACTACAAGACCACGCCT TISGLKPGVDY CCCGTGCTGGACTCCGACGG TITVYAVTLTKCTCCTTCTTCCTCTACAGCA SQLIHYMPISI AGCTCACCGTGGACAAGAGC NYRTEIAGGTGGCAGCAGGGGAACGT (SEQ ID NO: CTTCTCATGCTCCGTGATGC 252)ATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTC CCTGTCTCCCGGCGCCGGAGGCGGCGGATCCGGTGGAGTT TCTGATGTGCCGCGCGACCT GGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGC TGGTCTCTTCCTTATGCTGG TCATCTAAACTATTACCGCATCACTTACGGCGAAACAGGA GGCAATAGCCCTGTCCAGGA GTTCACTGTGCCTGGTCGTGGTGTGACAGCTACCATCAGC GGCCTTAAACCTGGCGTTGA TTATACCATCACTGTGTATGCTGTCACTCTGACTAAGTCT CAGCTGATACATTACATGCC AATTTCCATTAATTACCGGACCGAAATC (SEQ ID NO: 284) PRD- GVSDVPRDLEV GVSDVPRDLEVV EPKSSDKTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1171 VAATPTSLLIS AATPTSLLISWT (SEQ IDLGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WTLPHAGRAHY LPHAGRAHYYRI NO: 182)KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG YRITYGETGGN TYGETGGNSPVQCVVVDVSHEDPEV ATCTCCTGGACACTGCCTCA SPVQEFTVPGR EFTVPGRGVTATKFNWYVDGVEVHN CGCTGGCCGGGCTCATTACT GVTATISGLKP ISGLKPGVDYTIAKTKPREEQYNST ATAGAATTACCTACGGGGAG GVDYTITVYAV TVYAVTVTTTKVYRVVSVLTVLHQD ACAGGCGGGAACTCTCCCGT TVTTTKVIHYK IHYKPISINYRTWLNGKEYKCKVSN GCAGGAATTCACCGTGCCTG PISINYRTEIE EI (SEQ ID KALPAPIEKTISKGAAGGGGCGTGACTGCCACC PKSSDKTHTCP NO: 275) AKGQPREPQVYTLATCAGTGGGCTGAAGCCAGG PCPAPELLGGP PPSRDELTKNQVS AGTGGACTACACAATTACCGSVFLFPPKPKD LTCLVKGFYPSDI TGTACGCTGTGACTGTGACC TLMISRTPEVT AVEWESNGQPENNACAACTAAAGTGATCCACTA CVVVDVSHEDP YKTTPPVLDSDGS CAAACCCATCTCTATTAATTEVKFNWYVDGV FFLYSKLTVDKSR ATCGGACCGAAATTGAGCCT EVHNAKTKPRE WQQGNVFSCSVMHAAGAGCTCCGACAAAACCCA EQYNSTYRVVS EALHNHYTQKSLS CACATGCCCACCTTGTCCAGVLTVLHQDWLN LSPGK (SEQ CCCCCGAACTGCTGGGCGGC GKEYKCKVSNK ID NO: 283)CCTTCAGTCTTCCTCTTCCC ALPAPIEKTIS CCCAAAACCCAAGGACACCC KAKGQPREPQVTCATGATCTCCCGGACCCCT YTLPPSRDELT GAGGTCACATGCGTGGTGGT KNQVSLTCLVKGGACGTGAGCCACGAAGACC GFYPSDIAVEW CTGAGGTCAAGTTCAACTGG ESNGQPENNYKTACGTGGACGGCGTGGAGGT TTPPVLDSDGS GCATAATGCCAAGACAAAGC FFLYSKLTVDKCGCGGGAGGAGCAGTACAAC SRWQQGNVFSC AGCACGTACCGTGTGGTCAG SVMHEALHNHYCGTCCTCACCGTCCTGCACC TQKSLSLSPGK AGGACTGGCTGAATGGCAAG (SEQ ID NO:GAGTACAAGTGCAAGGTCTC 253) CAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTT CTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC ATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCCGGGAAA (SEQ ID NO: 285) PRD- GVSDVPRDLEVGVSDVPRDLEVV D KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1173 VAATPTSLLISAATPTSLLISWT LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WTLPHAGRAHY LPHAGRAHYYRIKDTLMISRTPEVT CTACCCCCACAAGCTTGCTG YRITYGETGGN TYGETGGNSPVQCVVVDVSHEDPEV ATCTCCTGGACACTGCCTCA SPVQEFTVPGR EFTVPGRGVTATKFNWYVDGVEVHN CGCTGGCCGGGCTCATTACT GVTATISGLKP ISGLKPGVDYTIAKTKPREEQYNST ATAGAATTACCTACGGGGAG GVDYTITVYAV TVYAVTVTTTKVYRVVSVLTVLHQD ACAGGCGGGAACTCTCCCGT TVTTTKVIHYK IHYKPISINYRTWLNGKEYKCKVSN GCAGGAATTCACCGTGCCTG PISINYRTEID EI (SEQ ID KALPAPIEKTISKGAAGGGGCGTGACTGCCACC KTHTCPPCPAP NO: 275) AKGQPREPQVYTLATCAGTGGGCTGAAGCCAGG ELLGGPSVFLF PPSRDELTKNQVS AGTGGACTACACAATTACCGPPKPKDTLMIS LTCLVKGFYPSDI TGTACGCTGTGACTGTGACC RTPEVTCVVVD AVEWESNGQPENNACAACTAAAGTGATCCACTA VSHEDPEVKFN YKTTPPVLDSDGS CAAACCCATCTCTATTAATTWYVDGVEVHNA FFLYSKLTVDKSR ATCGGACCGAAATTGACAAG KTKPREEQYNS WQQGNVFSCSVMHACCCACACATGCCCACCTTG TYRVVSVLTVL EALHNHYTQKSLS TCCAGCCCCCGAGCTGCTGGHQDWLNGKEYK LSPGK (SEQ GCGGCCCTTCAGTCTTCCTC CKVSNKALPAP ID NO: 283)TTCCCCCCAAAACCCAAGGA IEKTISKAKGQ CACCCTCATGATCTCCCGGA PREPQVYTLPPCCCCTGAGGTCACATGCGTG SRDELTKNQVS GTGGTGGACGTGAGCCACGA LTCLVKGFYPSAGACCCTGAGGTCAAGTTCA DIAVEWESNGQ ACTGGTACGTGGACGGCGTG PENNYKTTPPVGAGGTGCATAATGCCAAGAC LDSDGSFFLYS AAAGCCGCGGGAGGAGCAGT KLTVDKSRWQQACAACAGCACGTACCGTGTG GNVFSCSVMHE GTCAGCGTCCTCACCGTCCT ALHNHYTQKSLGCACCAGGACTGGCTGAATG SLSPGK GCAAGGAGTACAAGTGCAAG (SEQ ID NO:GTCTCCAACAAAGCCCTCCC 254) AGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA CACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTC CCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA GCTCACCGTGGACAAGAGCA GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA (SEQ ID NO: 286) PRD- GVSDVPRDLEV GVSDVPRDLEVV ESPKAQASSKTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1174 VAATPTSLLIS AATPTSLLISWTVPTAQPQAE LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WTLPHAGRAHY LPHAGRAHYYRIGLA (SEQ KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG YRITYGETGGN TYGETGGNSPVQID NO: CVVVDVSHEDPEV ATCTCCTGGACACTGCCTCA SPVQEFTVPGR EFTVPGRGVTAT 183)KFNWYVDGVEVHN CGCTGGCCGGGCTCATTACT GVTATISGLKP ISGLKPGVDYTIAKTKPREEQYNST ATAGAATTACCTACGGGGAG GVDYTITVYAV TVYAVTVTTTKVYRVVSVLTVLHQD ACAGGCGGGAACTCTCCCGT TVTTTKVIHYK IHYKPISINYRTWLNGKEYKCKVSN GCAGGAATTCACCGTGCCTG PISINYRTEIE EI (SEQ ID KALPAPIEKTISKGAAGGGGCGTGACTGCCACC SPKAQASSVPT NO: 275) AKGQPREPQVYTLATCAGTGGGCTGAAGCCAGG AQPQAEGLAKT PPSRDELTKNQVS AGTGGACTACACAATTACCGHTCPPCPAPEL LTCLVKGFYPSDI TGTACGCTGTGACTGTGACC LGGPSVFLFPP AVEWESNGQPENNACAACTAAAGTGATCCACTA KPKDTLMISRT YKTTPPVLDSDGS CAAACCCATCTCTATTAATTPEVTCVVVDVS FFLYSKLTVDKSR ATCGGACCGAAATTGAGTCT HEDPEVKFNWY WQQGNVFSCSVMHCCAAAGGCTCAGGCCAGCTC VDGVEVHNAKT EALHNHYTQKSLS CGTGCCTACCGCTCAGCCACKPREEQYNSTY LSPGK (SEQ AGGCTGAGGGCCTGGCTAAG RVVSVLTVLHQ ID NO: 283)ACCCACACATGCCCCCCTTG DWLNGKEYKCK TCCAGCTCCCGAACTGCTGG VSNKALPAPIEGCGGGCCTTCAGTCTTCCTC KTISKAKGQPR TTCCCCCCAAAACCCAAGGA EPQVYTLPPSRCACCCTCATGATCTCCCGGA DELTKNQVSLT CCCCTGAGGTCACATGCGTG CLVKGFYPSDIGTGGTGGACGTGAGCCACGA AVEWESNGQPE AGACCCTGAGGTCAAGTTCA NNYKTTPPVLDACTGGTACGTGGACGGCGTG SDGSFFLYSKL GAGGTGCATAATGCCAAGAC TVDKSRWQQGNAAAGCCGCGGGAGGAGCAGT VFSCSVMHEAL ACAACAGCACGTACCGTGTG HNHYTQKSLSLGTCAGCGTCCTCACCGTCCT SPGK (SEQ GCACCAGGACTGGCTGAATG ID NO: 255)GCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTA CACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAA GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCCGGGAAA (SEQ ID NO: 287) PRD- DKTHTCPPCPADKTHTCPPCPAP GAGGGGSG GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1175PELLGGPSVFL ELLGGPSVFLFP (SEQ ID ATPTSLLISWTLP ACCGTGCCCAGCACCTGAACFPPKPKDTLMI PKPKDTLMISRT NO: 181) HAGRAHYYRITYG TCCTGGGGGGACCGTCAGTCSRTPEVTCVVV PEVTCVVVDVSH ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRPGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTTTKVIHYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 275)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPGAGGGG 276)CCCGGGATGAGCTGACCAAG SGGVSDVPRDL AACCAGGTCAGCCTGACCTG EVVAATPTSLLCCTGGTCAAAGGCTTCTATC ISWTLPHAGRA CCAGCGACATCGCCGTGGAG HYYRITYGETGTGGGAGAGCAATGGGCAGCC GNSPVQEFTVP GGAGAACAACTACAAGACCA GRGVTATISGLCGCCTCCCGTGTTGGACTCC KPGVDYTITVY GACGGCTCCTTCTTCCTCTA AVTVTTTKVIHCAGCAAGCTCACCGTGGACA YKPISINYRTE AGAGCAGGTGGCAGCAGGGG I (SEQ IDAACGTCTTCTCATGCTCCGT NO: 256) GATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGCGC CGGAGGCGGCGGATCCGGTG GCGTGTCCGACGTGCCCCGGGATCTAGAAGTGGTGGCTGC TACCCCCACAAGCTTGCTGA TCTCCTGGACACTGCCTCACGCTGGCCGGGCTCATTACTA TAGAATTACCTACGGGGAGA CAGGCGGGAACTCTCCCGTGCAGGAATTCACCGTGCCTGG AAGGGGCGTGACTGCCACCA TCAGTGGGCTGAAGCCAGGAGTGGACTACACAATTACCGT GTACGCTGTGACTGTGACCA CAACTAAAGTGATCCACTACAAACCCATCTCTATTAATTA TCGGACCGAAATC (SEQ ID NO: 288) PRD- DKTHTCPPCPADKTHTCPPCPAP ELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1177PELLGGPSVFL ELLGGPSVFLFP EAQDGELD ATPTSLLISWTLP ACCGTGCCCAGCACCTGAACFPPKPKDTLMI PKPKDTLMISRT (SEQ ID HAGRAHYYRITYG TCCTGGGGGGACCGTCAGTCSRTPEVTCVVV PEVTCVVVDVSH NO: 184) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACCDVSHEDPEVKF EDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHNGVEVHNAKTKPR PGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTTTKVIHYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 275)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WTLPHAGRAHYTGGGAGAGCAATGGGCAGCC YRITYGETGGN GGAGAACAACTACAAGACCA SPVQEFTVPGRCGCCTCCCGTGCTGGACTCC GVTATISGLKP GACGGCTCCTTCTTCCTCTA GVDYTITVYAVCAGCAAGCTCACCGTGGACA TVTTTKVIHYK AGAGCAGGTGGCAGCAGGGG PISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 257)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCTCCTGGACACT GCCTCACGCTGGCCGGGCTCATTACTATAGAATTACCTAC GGGGAGACAGGCGGGAACTC TCCCGTGCAGGAATTCACCGTGCCTGGAAGGGGCGTGACT GCCACCATCAGTGGGCTGAA GCCAGGAGTGGACTACACAATTACCGTGTACGCTGTGACT GTGACCACAACTAAAGTGAT CCACTACAAACCCATCTCTATTAATTATCGGACCGAAATT (SEQ ID NO: 289) PRD- DKTHTCPPCPA DKTHTCPPCPAPGQPDEPGGS GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1178 PELLGGPSVFLELLGGPSVFLFP (SEQ ID ATPTSLLISWTLP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT NO: 185) HAGRAHYYRITYG TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKF EDPEVKFNWYVDPGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRPGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTTTKVIHYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 275)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPGQPDEP 276)CCCGGGATGAGCTGACCAAG GGSGVSDVPRD AACCAGGTCAGCCTGACCTG LEVVAATPTSLCCTGGTCAAAGGCTTCTATC LISWTLPHAGR CCAGCGACATCGCCGTGGAG AHYYRITYGETTGGGAGAGCAATGGGCAGCC GGNSPVQEFTV GGAGAACAACTACAAGACCA PGRGVTATISGCGCCTCCCGTGCTGGACTCC LKPGVDYTITV GACGGCTCCTTCTTCCTCTA YAVTVTTTKVICAGCAAGCTCACCGTGGACA HYKPISINYRT AGAGCAGGTGGCAGCAGGGG EI (SEQ IDAACGTCTTCTCATGCTCCGT NO: 258) GATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGCCA GCCCGACGAGCCTGGCGGGA GCGGCGTGAGCGACGTGCCACGGGATCTAGAAGTGGTGGC TGCTACCCCCACAAGCTTGC TGATCTCCTGGACACTGCCTCACGCTGGCCGGGCTCATTA CTATAGAATTACCTACGGGG AGACAGGCGGGAACTCTCCCGTGCAGGAATTCACCGTGCC TGGAAGGGGCGTGACTGCCA CCATCAGTGGGCTGAAGCCAGGAGTGGACTACACAATTAC CGTGTACGCTGTGACTGTGA CCACAACTAAAGTGATCCACTACAAACCCATCTCTATTAA TTATCGGACCGAAATT (SEQ ID NO: 290) PRD- DKTHTCPPCPADKTHTCPPCPAP GGSGSGSGS GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1180PELLGGPSVFL ELLGGPSVFLFP GSGS ATPTSLLISWTLP ACCGTGCCCAGCACCTGAACFPPKPKDTLMI PKPKDTLMISRT (SEQ ID HAGRAHYYRITYG TCCTGGGGGGACCGTCAGTCSRTPEVTCVVV PEVTCVVVDVSH NO: 186) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACCDVSHEDPEVKF EDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHNGVEVHNAKTKPR PGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTTTKVIHYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 275)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPGGSGSG 276)CCCGGGATGAGCTGACCAAG SGSGSGSGVSD AACCAGGTCAGCCTGACCTG VPRDLEVVAATCCTGGTCAAAGGCTTCTATC PTSLLISWTLP CCAGCGACATCGCCGTGGAG HAGRAHYYRITTGGGAGAGCAATGGGCAGCC YGETGGNSPVQ GGAGAACAACTACAAGACCA EFTVPGRGVTACGCCTCCCGTGCTGGACTCC TISGLKPGVDY GACGGCTCCTTCTTCCTCTA TITVYAVTVTTCAGCAAGCTCACCGTGGACA TKVIHYKPISI AGAGCAGGTGGCAGCAGGGG NYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 259)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGGCGG CAGCGGGTCTGGATCTGGCAGTGGGAGCGGCTCTGGCGTG AGCGACGTGCCACGGGATCT AGAAGTGGTGGCTGCTACCCCCACAAGCTTGCTGATCTCC TGGACACTGCCTCACGCTGG CCGGGCTCATTACTATAGAATTACCTACGGGGAGACAGGC GGGAACTCTCCCGTGCAGGA ATTCACCGTGCCTGGAAGGGGCGTGACTGCCACCATCAGT GGGCTGAAGCCAGGAGTGGA CTACACAATTACCGTGTACGCTGTGACTGTGACCACAACT AAAGTGATCCACTACAAACC CATCTCTATTAATTATCGGACCGAAATT (SEQ ID NO: 291) PRD- DKTHTCPPCPA DKTHTCPPCPAP ELQLEESAAGVSDVPRDLEVVA GACAAAACTCACACATGCCC 1284 PELLGGPSVFL ELLGGPSVFLFPEAQDGELD ATPTSLLISWDAP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMI PKPKDTLMISRT(SEQ ID RGLARYYRITYGE TCCTGGGGGGACCGTCAGTC SRTPEVTCVVV PEVTCVVVDVSHNO: 184) TGGNSPVQEFTVF TTCCTCTTCCCCCCAAAACC DVSHEDPEVKF EDPEVKFNWYVDGRGTTATISGLKP CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRGVDYTITVYAVTI CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSDRDGTRSFDPISI TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG NYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 277)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WDAPRGLARYYTGGGAGAGCAATGGGCAGCC RITYGETGGNS GGAGAACAACTACAAGACCA PVQEFTVFGRGCGCCTCCCGTGCTGGACTCC TTATISGLKPG GACGGCTCCTTCTTCCTCTA VDYTITVYAVTCAGCAAGCTCACCGTGGACA IDRDGTRSFDP AGAGCAGGTGGCAGCAGGGG ISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 260)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGGACGC TCCGAGAGGTCTGGCTCGATATTACCGCATCACTTACGGC GAAACAGGAGGCAATAGCCC TGTCCAGGAGTTCACTGTGTTCGGTCGTGGTACCACAGCT ACCATCAGCGGCCTTAAACC TGGCGTTGATTATACCATCACTGTGTATGCTGTCACTATC GACCGTGACGGTACCCGCAG CTTCGACCCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 292) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1285 PELLGGPSVFLELLGGPSVFLFP EAQDGELD ATPTSLLISWDAP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID AGLARYYRITYGE TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 184) TGGNSPVQEFTVV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD GRGNTATISGLKP CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRGVDYTITVYAVTI CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSFRDGPVTWDPISI TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG NYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 278)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WDAPAGLARYYTGGGAGAGCAATGGGCAGCC RITYGETGGNS GGAGAACAACTACAAGACCA PVQEFTVVGRGCGCCTCCCGTGCTGGACTCC NTATISGLKPG GACGGCTCCTTCTTCCTCTA VDYTITVYAVTCAGCAAGCTCACCGTGGACA IFRDGPVTWDP AGAGCAGGTGGCAGCAGGGG ISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 261)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGGACGC TCCGGCTGGTCTGGCTCGATATTACCGCATCACTTACGGC GAAACAGGAGGCAATAGCCC TGTCCAGGAGTTCACTGTGGTCGGTCGTGGTAACACAGCT ACCATCAGCGGCCTTAAACC TGGCGTTGATTATACCATCACTGTGTATGCTGTCACTATC TTCCGTGACGGTCCCGTCAC CTGGGACCCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 293) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1286 PELLGGPSVFLELLGGPSVFLFP EAQDGELD ATPTSLLISWDAP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID KGLARYYRITYGE TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 184) TGGNSPVQEFTVV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD GRGNTATISGLKP CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRGVDYTITVYAVTI CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSFRDGPVTWDPISI TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG NYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 279)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WDAPKGLARYYTGGGAGAGCAATGGGCAGCC RITYGETGGNS GGAGAACAACTACAAGACCA PVQEFTVVGRGCGCCTCCCGTGCTGGACTCC NTATISGLKPG GACGGCTCCTTCTTCCTCTA VDYTITVYAVTCAGCAAGCTCACCGTGGACA IFRDGPVTWDP AGAGCAGGTGGCAGCAGGGG ISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 262)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGGACGC TCCGAAGGGTCTGGCTCGATATTACCGCATCACTTACGGC GAAACAGGAGGCAATAGCCC TGTCCAGGAGTTCACTGTGGTCGGTCGTGGTAACACAGCT ACCATCAGCGGCCTTAAACC TGGCGTTGATTATACCATCACTGTGTATGCTGTCACTATC TTCCGTGACGGTCCCGTCAC CTGGGACCCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 294) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1287 PELLGGPSVFLELLGGPSVFLFP EAQDGELD ATPTSLLISWSLP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID NPGNAHYYRITYG TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 184) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRPGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTDTGFITYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 280)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WSLPNPGNAHYTGGGAGAGCAATGGGCAGCC YRITYGETGGN GGAGAACAACTACAAGACCA SPVQEFTVPGRCGCCTCCCGTGCTGGACTCC GVTATISGLKP GACGGCTCCTTCTTCCTCTA GVDYTITVYAVCAGCAAGCTCACCGTGGACA TVTDTGFITYK AGAGCAGGTGGCAGCAGGGG PISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 263)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGTCTCT GCCGAATCCGGGTAACGCCCATTATTACCGCATCACTTAC GGCGAAACAGGAGGCAATAG CCCTGTCCAGGAGTTCACTGTGCCTGGTCGTGGTGTTACA GCTACCATCAGCGGCCTTAA ACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACT GTTACTGACACAGGTTTCAT CACGTACAAACCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 295) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1288 PELLGGPSVFLELLGGPSVFLFP EAQDGELD ATPTSLLISWSLP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID HQGKANYYRITYG TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 184) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRPGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTDTGYLKYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 281)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQDGELD AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WSLPHQGKANYTGGGAGAGCAATGGGCAGCC YRITYGETGGN GGAGAACAACTACAAGACCA SPVQEFTVPGRCGCCTCCCGTGCTGGACTCC GVTATISGLKP GACGGCTCCTTCTTCCTCTA GVDYTITVYAVCAGCAAGCTCACCGTGGACA TVTDTGYLKYK AGAGCAGGTGGCAGCAGGGG PISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 264)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGACGGAGAA CTGGATGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGTCTCT GCCGCACCAAGGTAAAGCCAATTATTACCGCATCACTTAC GGCGAAACAGGAGGCAATAG CCCTGTCCAGGAGTTCACTGTGCCTGGTCGTGGTGTTACA GCTACCATCAGCGGCCTTAA ACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACT GTTACTGATACAGGGTACCT CAAGTACAAACCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 296) PRD- GVSDVPRDLEV GVSDVPRDLEVVEPKSSD KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1301 VAATPTSLLIS AATPTSLLISWD(SEQ ID LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WDAPRGLARYY APRGLARYYRITNO: 182) KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG RITYGETGGNS YGETGGNSPVQECVVVDVSHEDPEV ATCAGCTGGGACGCTCCGAG PVQEFTVFGRG FTVFGRGTTATIKFNWYVDGVEVHN AGGTCTGGCTCGATATTACC TTATISGLKPG SGLKPGVDYTITAKTKPREEQYNST GCATCACTTACGGCGAAACA VDYTITVYAVT VYAVTIDRDGTRYRVVSVLTVLHQD GGAGGCAATAGCCCTGTCCA IDRDGTRSFDP SFDPISINYRTEWLNGKEYKCKVSN GGAGTTCACTGTGTTCGGTC ISINYRTEIEP I (SEQ ID KALPAPIEKTISKGTGGTACCACAGCTACCATC KSSDKTHTCPP NO: 277) AKGQPREPQVYTLAGCGGCCTTAAACCTGGCGT CPAPELLGGPS PPSRDELTKNQVS TGATTATACCATCACTGTGTVFLFPPKPKDT LTCLVKGFYPSDI ATGCTGTCACTATCGACCGT LMISRTPEVTC AVEWESNGQPENNGACGGTACCCGCAGCTTCGA VVVDVSHEDPE YKTTPPVLDSDGS CCCAATTTCCATTAATTACCVKFNWYVDGVE FFLYSKLTVDKSR GGACCGAAATTGAGCCTAAG VHNAKTKPREE WQQGNVFSCSVMHAGCTCCGACAAAACCCACAC QYNSTYRVVSV EALHNHYTQKSLS ATGCCCACCTTGTCCAGCCCLTVLHQDWLNG LSPGK (SEQ CCGAACTGCTGGGCGGCCCT KEYKCKVSNKA ID NO: 283)TCAGTCTTCCTCTTCCCCCC LPAPIEKTISK AAAACCCAAGGACACCCTCA AKGQPREPQVYTGATCTCCCGGACCCCTGAG TLPPSRDELTK GTCACATGCGTGGTGGTGGA NQVSLTCLVKGCGTGAGCCACGAAGACCCTG FYPSDIAVEWE AGGTCAAGTTCAACTGGTAC SNGQPENNYKTGTGGACGGCGTGGAGGTGCA TPPVLDSDGSF TAATGCCAAGACAAAGCCGC FLYSKLTVDKSGGGAGGAGCAGTACAACAGC RWQQGNVFSCS ACGTACCGTGTGGTCAGCGT VMHEALHNHYTCCTCACCGTCCTGCACCAGG QKSLSLSPGK ACTGGCTGAATGGCAAGGAG (SEQ ID NO:TACAAGTGCAAGGTCTCCAA 265) CAAAGCCCTCCCAGCCCCCA TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC CGGGAAA (SEQ ID NO: 297) PRD- GVSDVPRDLEVGVSDVPRDLEVV EPKSSD KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1302 VAATPTSLLISAATPTSLLISWD (SEQ ID LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WDAPAGLARYYAPAGLARYYRIT NO: 182) KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG RITYGETGGNSYGETGGNSPVQE CVVVDVSHEDPEV ATCAGCTGGGACGCTCCGGC PVQEFTVVGRG FTVVGRGNTATIKFNWYVDGVEVHN TGGTCTGGCTCGATATTACC NTATISGLKPG SGLKPGVDYTITAKTKPREEQYNST GCATCACTTACGGCGAAACA VDYTITVYAVT VYAVTIFRDGPVYRVVSVLTVLHQD GGAGGCAATAGCCCTGTCCA IFRDGPVTWDP TWDPISINYRTEWLNGKEYKCKVSN GGAGTTCACTGTGGTCGGTC ISINYRTEIEP I (SEQ ID KALPAPIEKTISKGTGGTAACACAGCTACCATC KSSDKTHTCPP NO: 278) AKGQPREPQVYTLAGCGGCCTTAAACCTGGCGT CPAPELLGGPS PPSRDELTKNQVS TGATTATACCATCACTGTGTVFLFPPKPKDT LTCLVKGFYPSDI ATGCTGTCACTATCTTCCGT LMISRTPEVTC AVEWESNGQPENNGACGGTCCCGTCACCTGGGA VVVDVSHEDPE YKTTPPVLDSDGS CCCAATTTCCATTAATTACCVKFNWYVDGVE FFLYSKLTVDKSR GGACCGAAATTGAGCCTAAG VHNAKTKPREE WQQGNVFSCSVMHAGCTCCGACAAAACCCACAC QYNSTYRVVSV EALHNHYTQKSLS ATGCCCACCTTGTCCAGCCCLTVLHQDWLNG LSPGK (SEQ CCGAACTGCTGGGCGGCCCT KEYKCKVSNKA ID NO: 283)TCAGTCTTCCTCTTCCCCCC LPAPIEKTISK AAAACCCAAGGACACCCTCA AKGQPREPQVYTGATCTCCCGGACCCCTGAG TLPPSRDELTK GTCACATGCGTGGTGGTGGA NQVSLTCLVKGCGTGAGCCACGAAGACCCTG FYPSDIAVEWE AGGTCAAGTTCAACTGGTAC SNGQPENNYKTGTGGACGGCGTGGAGGTGCA TPPVLDSDGSF TAATGCCAAGACAAAGCCGC FLYSKLTVDKSGGGAGGAGCAGTACAACAGC RWQQGNVFSCS ACGTACCGTGTGGTCAGCGT VMHEALHNHYTCCTCACCGTCCTGCACCAGG QKSLSLSPGK ACTGGCTGAATGGCAAGGAG (SEQ ID NO:TACAAGTGCAAGGTCTCCAA 266) CAAAGCCCTCCCAGCCCCCA TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC CGGGAAA (SEQ ID NO: 298) PRD- GVSDVPRDLEVGVSDVPRDLEVV EPKSSD KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1303 VAATPTSLLISAATPTSLLISWD (SEQ ID LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WDAPKGLARYYAPKGLARYYRIT NO: 182) KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG RITYGETGGNSYGETGGNSPVQE CVVVDVSHEDPEV ATCAGCTGGGACGCTCCGAA PVQEFTVVGRG FTVVGRGNTATIKFNWYVDGVEVHN GGGTCTGGCTCGATATTACC NTATISGLKPG SGLKPGVDYTITAKTKPREEQYNST GCATCACTTACGGCGAAACA VDYTITVYAVT VYAVTIFRDGPVYRVVSVLTVLHQD GGAGGCAATAGCCCTGTCCA IFRDGPVTWDP TWDPISINYRTEWLNGKEYKCKVSN GGAGTTCACTGTGGTCGGTC ISINYRTEIEP I (SEQ ID KALPAPIEKTISKGTGGTAACACAGCTACCATC KSSDKTHTCPP NO: 279) AKGQPREPQVYTLAGCGGCCTTAAACCTGGCGT CPAPELLGGPS PPSRDELTKNQVS TGATTATACCATCACTGTGTVFLFPPKPKDT LTCLVKGFYPSDI ATGCTGTCACTATCTTCCGT LMISRTPEVTC AVEWESNGQPENNGACGGTCCCGTCACCTGGGA VVVDVSHEDPE YKTTPPVLDSDGS CCCAATTTCCATTAATTACCVKFNWYVDGVE FFLYSKLTVDKSR GGACCGAAATTGAGCCTAAG VHNAKTKPREE WQQGNVFSCSVMHAGCTCCGACAAAACCCACAC QYNSTYRVVSV EALHNHYTQKSLS ATGCCCACCTTGTCCAGCCCLTVLHQDWLNG LSPGK (SEQ CCGAACTGCTGGGCGGCCCT KEYKCKVSNKA ID NO: 283)TCAGTCTTCCTCTTCCCCCC LPAPIEKTISK AAAACCCAAGGACACCCTCA AKGQPREPQVYTGATCTCCCGGACCCCTGAG TLPPSRDELTK GTCACATGCGTGGTGGTGGA NQVSLTCLVKGCGTGAGCCACGAAGACCCTG FYPSDIAVEWE AGGTCAAGTTCAACTGGTAC SNGQPENNYKTGTGGACGGCGTGGAGGTGCA TPPVLDSDGSF TAATGCCAAGACAAAGCCGC FLYSKLTVDKSGGGAGGAGCAGTACAACAGC RWQQGNVFSCS ACGTACCGTGTGGTCAGCGT VMHEALHNHYTCCTCACCGTCCTGCACCAGG QKSLSLSPGK ACTGGCTGAATGGCAAGGAG (SEQ ID NO:TACAAGTGCAAGGTCTCCAA 267) CAAAGCCCTCCCAGCCCCCA TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC CGGGAAA (SEQ ID NO: 299) PRD- GVSDVPRDLEVGVSDVPRDLEVV EPKSSD KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1304 VAATPTSLLISAATPTSLLISWS (SEQ ID LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WSLPNPGNAHYLPNPGNAHYYRI NO: 182) KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG YRITYGETGGNTYGETGGNSPVQ CVVVDVSHEDPEV ATCAGCTGGTCTCTGCCGAA SPVQEFTVPGR EFTVPGRGVTATKFNWYVDGVEVHN TCCGGGTAACGCCCATTATT GVTATISGLKP ISGLKPGVDYTIAKTKPREEQYNST ACCGCATCACTTACGGCGAA GVDYTITVYAV TVYAVTVTDTGFYRVVSVLTVLHQD ACAGGAGGCAATAGCCCTGT TVTDTGFITYK ITYKPISINYRTWLNGKEYKCKVSN CCAGGAGTTCACTGTGCCTG PISINYRTEIE EI (SEQ ID KALPAPIEKTISKGTCGTGGTGTTACAGCTACC PKSSDKTHTCP NO: 280) AKGQPREPQVYTLATCAGCGGCCTTAAACCTGG PCPAPELLGGP PPSRDELTKNQVS CGTTGATTATACCATCACTGSVFLFPPKPKD LTCLVKGFYPSDI TGTATGCTGTCACTGTTACT TLMISRTPEVT AVEWESNGQPENNGACACAGGTTTCATCACGTA CVVVDVSHEDP YKTTPPVLDSDGS CAAACCAATTTCCATTAATTEVKFNWYVDGV FFLYSKLTVDKSR ACCGGACCGAAATTGAGCCT EVHNAKTKPRE WQQGNVFSCSVMHAAGAGCTCCGACAAAACCCA EQYNSTYRVVS EALHNHYTQKSLS CACATGCCCACCTTGTCCAGVLTVLHQDWLN LSPGK (SEQ CCCCCGAACTGCTGGGCGGC GKEYKCKVSNK ID NO: 283)CCTTCAGTCTTCCTCTTCCC ALPAPIEKTIS CCCAAAACCCAAGGACACCC KAKGQPREPQVTCATGATCTCCCGGACCCCT YTLPPSRDELT GAGGTCACATGCGTGGTGGT KNQVSLTCLVKGGACGTGAGCCACGAAGACC GFYPSDIAVEW CTGAGGTCAAGTTCAACTGG ESNGQPENNYKTACGTGGACGGCGTGGAGGT TTPPVLDSDGS GCATAATGCCAAGACAAAGC FFLYSKLTVDKCGCGGGAGGAGCAGTACAAC SRWQQGNVFSC AGCACGTACCGTGTGGTCAG SVMHEALHNHYCGTCCTCACCGTCCTGCACC TQKSLSLSPGK AGGACTGGCTGAATGGCAAG (SEQ ID NO:GAGTACAAGTGCAAGGTCTC 268) CAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTT CTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC ATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCCGGGAAA (SEQ ID NO: 300) PRD- GVSDVPRDLEVGVSDVPRDLEVV EPKSSD KTHTCPPCPAPEL GGCGTGAGCGACGTGCCCCG 1305 VAATPTSLLISAATPTSLLISWS (SEQ ID LGGPSVFLFPPKP GGATCTAGAAGTGGTGGCTG WSLPHQGKANYLPHQGKANYYRI NO: 182) KDTLMISRTPEVT CTACCCCCACAAGCTTGCTG YRITYGETGGNTYGETGGNSPVQ CVVVDVSHEDPEV ATCAGCTGGTCTCTGCCGCA SPVQEFTVPGR EFTVPGRGVTATKFNWYVDGVEVHN CCAAGGTAAAGCCAATTATT GVTATISGLKP ISGLKPGVDYTIAKTKPREEQYNST ACCGCATCACTTACGGCGAA GVDYTITVYAV TVYAVTVTDTGYYRVVSVLTVLHQD ACAGGAGGCAATAGCCCTGT TVTDTGYLKYK LKYKPISINYRTWLNGKEYKCKVSN CCAGGAGTTCACTGTGCCTG PISINYRTEIE EI (SEQ ID KALPAPIEKTISKGTCGTGGTGTTACAGCTACC PKSSDKTHTCP NO: 281) AKGQPREPQVYTLATCAGCGGCCTTAAACCTGG PCPAPELLGGP PPSRDELTKNQVS CGTTGATTATACCATCACTGSVFLFPPKPKD LTCLVKGFYPSDI TGTATGCTGTCACTGTTACT TLMISRTPEVT AVEWESNGQPENNGATACAGGGTACCTCAAGTA CVVVDVSHEDP YKTTPPVLDSDGS CAAACCAATTTCCATTAATTEVKFNWYVDGV FFLYSKLTVDKSR ACCGGACCGAAATTGAGCCT EVHNAKTKPRE WQQGNVFSCSVMHAAGAGCTCCGACAAAACCCA EQYNSTYRVVS EALHNHYTQKSLS CACATGCCCACCTTGTCCAGVLTVLHQDWLN LSPGK (SEQ CCCCCGAACTGCTGGGCGGC GKEYKCKVSNK ID NO: 283)CCTTCAGTCTTCCTCTTCCC ALPAPIEKTIS CCCAAAACCCAAGGACACCC KAKGQPREPQVTCATGATCTCCCGGACCCCT YTLPPSRDELT GAGGTCACATGCGTGGTGGT KNQVSLTCLVKGGACGTGAGCCACGAAGACC GFYPSDIAVEW CTGAGGTCAAGTTCAACTGG ESNGQPENNYKTACGTGGACGGCGTGGAGGT TTPPVLDSDGS GCATAATGCCAAGACAAAGC FFLYSKLTVDKCGCGGGAGGAGCAGTACAAC SRWQQGNVFSC AGCACGTACCGTGTGGTCAG SVMHEALHNHYCGTCCTCACCGTCCTGCACC TQKSLSLSPGK AGGACTGGCTGAATGGCAAG (SEQ ID NO:GAGTACAAGTGCAAGGTCTC 269) CAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTT CTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC ATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCCGGGAAA (SEQ ID NO: 301) PRD- DKTHTCPPCPADKTHTCPPCPAP ELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1471PELLGGPSVFL ELLGGPSVFLFP EAQEGELE ATPTSLLISWTLP ACCGTGCCCAGCACCTGAACFPPKPKDTLMI PKPKDTLMISRT (SEQ ID HAGRAHYYRITYG TCCTGGGGGGACCGTCAGTCSRTPEVTCVVV PEVTCVVVDVSH NO: 187) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACCDVSHEDPEVKF EDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHNGVEVHNAKTKPR PGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTTTKVIHYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 275)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQEGELE AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WTLPHAGRAHYTGGGAGAGCAATGGGCAGCC YRITYGETGGN GGAGAACAACTACAAGACCA SPVQEFTVPGRCGCCTCCCGTGCTGGACTCC GVTATISGLKP GACGGCTCCTTCTTCCTCTA GVDYTITVYAVCAGCAAGCTCACCGTGGACA TVTTTKVIHYK AGAGCAGGTGGCAGCAGGGG PISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 270)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGAAGGAGAA CTGGAAGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCTCCTGGACACT GCCTCACGCTGGCCGGGCTCATTACTATAGAATTACCTAC GGGGAGACAGGCGGGAACTC TCCCGTGCAGGAATTCACCGTGCCTGGAAGGGGCGTGACT GCCACCATCAGTGGGCTGAA GCCAGGAGTGGACTACACAATTACCGTGTACGCTGTGACT GTGACCACAACTAAAGTGAT CCACTACAAACCCATCTCTATTAATTATCGGACCGAAATT (SEQ ID NO: 302) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1472 PELLGGPSVFLELLGGPSVFLFP EAQEGELE ATPTSLLISWDAP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID AGLARYYRITYGE TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 187) TGGNSPVQEFTVV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD GRGNTATISGLKP CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRGVDYTITVYAVTI CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSFRDGPVTWDPISI TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG NYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 278)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQEGELE AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WDAPAGLARYYTGGGAGAGCAATGGGCAGCC RITYGETGGNS GGAGAACAACTACAAGACCA PVQEFTVVGRGCGCCTCCCGTGCTGGACTCC NTATISGLKPG GACGGCTCCTTCTTCCTCTA VDYTITVYAVTCAGCAAGCTCACCGTGGACA IFRDGPVTWDP AGAGCAGGTGGCAGCAGGGG ISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 271)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGAAGGAGAA CTGGAAGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGGACGC TCCGGCTGGTCTGGCTCGATATTACCGCATCACTTACGGC GAAACAGGAGGCAATAGCCC TGTCCAGGAGTTCACTGTGGTCGGTCGTGGTAACACAGCT ACCATCAGCGGCCTTAAACC TGGCGTTGATTATACCATCACTGTGTATGCTGTCACTATC TTCCGTGACGGTCCCGTCAC CTGGGACCCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 303) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1473 PELLGGPSVFLELLGGPSVFLFP EAQEGELE ATPTSLLISWDAP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID KGLARYYRITYGE TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 187) TGGNSPVQEFTVV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD GRGNTATISGLKP CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRGVDYTITVYAVTI CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSFRDGPVTWDPISI TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG NYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 279)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQEGELE AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WDAPKGLARYYTGGGAGAGCAATGGGCAGCC RITYGETGGNS GGAGAACAACTACAAGACCA PVQEFTVVGRGCGCCTCCCGTGCTGGACTCC NTATISGLKPG GACGGCTCCTTCTTCCTCTA VDYTITVYAVTCAGCAAGCTCACCGTGGACA IFRDGPVTWDP AGAGCAGGTGGCAGCAGGGG ISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 272)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGAAGGAGAA CTGGAAGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGGACGC TCCGAAGGGTCTGGCTCGATATTACCGCATCACTTACGGC GAAACAGGAGGCAATAGCCC TGTCCAGGAGTTCACTGTGGTCGGTCGTGGTAACACAGCT ACCATCAGCGGCCTTAAACC TGGCGTTGATTATACCATCACTGTGTATGCTGTCACTATC TTCCGTGACGGTCCCGTCAC CTGGGACCCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 304) PRD- DKTHTCPPCPA DKTHTCPPCPAPELQLEESAA GVSDVPRDLEVVA GACAAAACTCACACATGCCC 1474 PELLGGPSVFLELLGGPSVFLFP EAQEGELE ATPTSLLISWSLP ACCGTGCCCAGCACCTGAAC FPPKPKDTLMIPKPKDTLMISRT (SEQ ID HQGKANYYRITYG TCCTGGGGGGACCGTCAGTC SRTPEVTCVVVPEVTCVVVDVSH NO: 187) ETGGNSPVQEFTV TTCCTCTTCCCCCCAAAACC DVSHEDPEVKFEDPEVKFNWYVD PGRGVTATISGLK CAAGGACACCCTCATGATCT NWYVDGVEVHN GVEVHNAKTKPRPGVDYTITVYAVT CCCGGACCCCTGAGGTCACA AKTKPREEQYN EEQYNSTYRVVSVTDTGYLKYKPIS TGCGTGGTGGTGGACGTGAG STYRVVSVLTV VLTVLHQDWLNG INYRTEI (SEQCCACGAAGACCCTGAGGTCA LHQDWLNGKEY KEYKCKVSNKAL ID NO: 281)AGTTCAACTGGTACGTGGAC KCKVSNKALPA PAPIEKTISKAK GGCGTGGAGGTGCATAATGCPIEKTISKAKG GQPREPQVYTLP CAAGACAAAGCCGCGGGAGG QPREPQVYTLP PSRDELTKNQVSAGCAGTACAACAGCACGTAC PSRDELTKNQV LTCLVKGFYPSD CGTGTGGTCAGCGTCCTCACSLTCLVKGFYP IAVEWESNGQPE CGTCCTGCACCAGGACTGGC SDIAVEWESNG NNYKTTPPVLDSTGAATGGCAAGGAGTACAAG QPENNYKTTPP DGSFFLYSKLTV TGCAAGGTCTCCAACAAAGCVLDSDGSFFLY DKSRWQQGNVFS CCTCCCAGCCCCCATCGAGA SKLTVDKSRWQ CSVMHEALHNHYAAACCATCTCCAAAGCCAAA QGNVFSCSVMH TQKSLSLSP GGGCAGCCCCGAGAACCACAEALHNHYTQKS (SEQ ID NO: GGTGTACACCCTGCCCCCAT LSLSPELQLEE 276)CCCGGGATGAGCTGACCAAG SAAEAQEGELE AACCAGGTCAGCCTGACCTG GVSDVPRDLEVCCTGGTCAAAGGCTTCTATC VAATPTSLLIS CCAGCGACATCGCCGTGGAG WSLPHQGKANYTGGGAGAGCAATGGGCAGCC YRITYGETGGN GGAGAACAACTACAAGACCA SPVQEFTVPGRCGCCTCCCGTGCTGGACTCC GVTATISGLKP GACGGCTCCTTCTTCCTCTA GVDYTITVYAVCAGCAAGCTCACCGTGGACA TVTDTGYLKYK AGAGCAGGTGGCAGCAGGGG PISINYRTEIAACGTCTTCTCATGCTCCGT (SEQ ID NO: GATGCATGAGGCTCTGCACA 273)ACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCCGAGCT GCAGCTGGAGGAAAGCGCCGCTGAGGCTCAGGAAGGAGAA CTGGAAGGCGTGAGCGACGT GCCACGGGATCTAGAAGTGGTGGCTGCTACCCCCACAAGC TTGCTGATCAGCTGGTCTCT GCCGCACCAAGGTAAAGCCAATTATTACCGCATCACTTAC GGCGAAACAGGAGGCAATAG CCCTGTCCAGGAGTTCACTGTGCCTGGTCGTGGTGTTACA GCTACCATCAGCGGCCTTAA ACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACT GTTACTGATACAGGGTACCT CAAGTACAAACCAATTTCCATTAATTACCGGACCGAAATT (SEQ ID NO: 305)

The SEQ ID NOs of exemplary leader (N-terminal extension) and C-terminaltail sequences of the invention are presented in Table 7.

TABLE 7 Summary of Exemplary Sequences SEQ ID NO Description NameSequence 306 Exemplary leader AdNT1 MGVSDVPRDL 307 Exemplary leaderAdNT2 GVSDVPRDL 308 Exemplary leader AdNT3 VSDVPRDL 309 Exemplary leaderAdNT4 SDVPRDL 310 Exemplary leader AdNT5 DVPRDL 311 Exemplary leaderAdNT6 VPRDL 312 Exemplary leader AdNT7 PRDL — Exemplary leader AdNT8 RDL— Exemplary leader AdNT9 DL 211 Exemplary tail AdCT1 EIDKPSQ —Exemplary tail AdCT2 EI 313 Exemplary tail AdCT3 EIEPKSS 314Exemplary tail AdCT4 EIDKPC 315 Exemplary tail AdCT5 EIDKP 316Exemplary tail AdCT6 EIDK 317 Exemplary tail AdCT7 EIDKPS 318Exemplary tail AdCT8 EIEKPSQ 319 Exemplary tail AdCT9 EIDKPSQLE 320Exemplary tail AdCT10 EIEDEDEDEDED 321 Exemplary tail AdCT11 EGSGS 322Exemplary tail AdCT12 EIDKPCQ 189 Exemplary tail AdCT13 GSGC 323Exemplary tail AdCT14 EGSGC 324 Exemplary tail AdCT15 EIDKPCQLE 325Exemplary tail AdCT16 EIDKPSQHHHHHH 326 Exemplary tail AdCT17 GSGCHHHHHH327 Exemplary tail AdCT18 EGSGCHHHHHH 328 Tag T1 HHHHHHIV. Nucleic Acid-Protein Fusion Technology

In one aspect, the invention provides an Adnectin comprising fibronectintype III domains that binds myostatin. One way to rapidly make and testFn3 domains with specific binding properties is the nucleic acid-proteinfusion technology of Adnexus, a Bristol-Myers Squibb R&D Company. Thisdisclosure utilizes the in vitro expression and tagging technology,termed ‘PROfusion’ which exploits nucleic acid-protein fusions (RNA- andDNA-protein fusions) to identify novel polypeptides and amino acidmotifs that are important for binding to proteins. Nucleic acid-proteinfusion technology is a technology that covalently couples a protein toits encoding genetic information. For a detailed description of theRNA-protein fusion technology and fibronectin-based scaffold proteinlibrary screening methods see Szostak et al., U.S. Pat. Nos. 6,258,558,6,261,804, 6,214,553, 6,281,344, 6,207,446, 6,518,018 and 6,818,418;Roberts et al., Proc. Natl. Acad. Sci., 1997; 94:12297-12302; and Kurzet al., Molecules, 2000; 5:1259-64, all of which are herein incorporatedby reference.

V. Vectors and Polynucleotides

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr. Purif., 26(I):96-105 (October2002); Connell, N. D., Curr. Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (September 1996);and Sharp et al., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. Generally, the DNA encoding thepolypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding site, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An exemplary N-terminal leader sequence forproduction of polypeptides in a mammalian system is:METDTLLLWVLLLWVPGSTG (SEQ ID NO: 177), which is removed by the host cellfollowing expression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., a yeast invertase leader, a factor leader (including Saccharomycesand Kluyveromyces alpha-factor leaders), or acid phosphatase leader, theC. albicans glucoamylase leader, or the signal sequence described inU.S. Pat. No. 5,631,144. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available. The DNA for such precursor regions maybe ligated in reading frame to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein of the invention, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein of the invention. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding protein of the invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, σ-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein of the invention.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO 94/11026 and the expressionvector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude, but are not limited to, a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York(1985)), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

VI. Protein Production

The present invention is also directed to cell lines that express ananti-myostatin Adnectin or fusion polypeptide thereof. Creation andisolation of cell lines producing an anti-myostatin Adnectin can beaccomplished using standard techniques known in the art, such as thosedescribed herein.

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.In the examples shown here, the host cells used for high-throughputprotein production (HTPP) and mid-scale production were those from theHMS 174-bacterial strain.

Adnectins of the present invention can also be obtained in aglycosylatedform by producing the Adnectins in, e.g., prokaryotic cells (e.g., E.coli). Notably, aglycosylated forms of the Adnectins of the inventionexhibit the same affinity, potency, and mechanism of action asglycosylated Adnectins when tested in vitro.

The host cells used to produce the proteins of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma)) aresuitable for culturing the host cells. In addition, many of the mediadescribed in Ham et al., Meth. Enzymol., 58:44 (1979), Barites et al.,Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866,4,927,762, 4,560,655, 5,122,469, 6,048,728, 5,672,502, or U.S. Pat. No.RE 30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as Gentamycin drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

Proteins of the invention can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2ndEdition, The Pierce Chemical Co., Rockford, Ill. (1984)). Modificationsto the protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrant distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, or preferablyat least 95% pure, and most preferably at least 98% pure. Regardless ofthe exact numerical value of the purity, the polypeptide is sufficientlypure for use as a pharmaceutical product.

VII. Biophysical and Biochemical Characterization

Binding of an anti-myostatin Adnectin of the invention to a targetmolecule (e.g., myostatin) may be assessed in terms of equilibriumconstants (e.g., dissociation, K_(D)) and in terms of kinetic constants(e.g., on-rate constant, k_(on) and off-rate constant, k_(off)). AnAdnectin will generally bind to a target molecule with a K_(D) of lessthan 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, or 100 pM, althoughhigher K_(D) values may be tolerated where the k_(off) is sufficientlylow or the k_(on), is sufficiently high.

In Vitro Assays for Binding Affinity

Anti-myostatin Adnectins that bind to and antagonize myostatin can beidentified using various in vitro assays. Preferably, the assays arehigh-throughput assays that allow for screening multiple candidateAdnectins simultaneously. In some embodiments, BMP-11, which shares 90%amino acid identity with myostatin, can be used as a surrogate formyostatin in in vitro assays when the assay is performed undersaturating conditions.

Notably, anti-myostatin Adnectins fused to Fc domains can bind bothmyostatin and BMP-11, whereas monoAdnectins bind only to myostatin.Without being bound by theory, this may reflect the increased avidity ofbivalent Fc-fused Adnectins compared to monovalent Adnectins. Similarenhanced binding to BMP11 is observed with bivalent PEGylated Adnectins,such as ATI-1341, which comprise Adnectins fused to two ends of a 20 kDaPEG moiety.

Exemplary assays for determining the binding affinity of anti-myostatinAdnectins are described in the Examples infra, and include, but are notlimited to, solution phase methods such as the kinetic exclusion assay(KinExA) (Blake et al., JBC 1996; 271:27677-85; Drake et al., AnalBiochem 2004; 328:35-43), surface plasmon resonance (SPR) with theBiacore system (Uppsala, Sweden) (Welford et al., Opt. Quant. Elect1991; 23:1; Morton and Myszka, Methods in Enzymology 1998; 295:268) andhomogeneous time resolved fluorescence (HTRF) assays (Newton et al., JBiomol Screen 2008; 13:674-82; Patel et al., Assay Drug Dev Technol2008; 6:55-68).

In some embodiments, biomolecular interactions can be monitored in realtime with the Biacore system, which uses SPR to detect changes in theresonance angle of light at the surface of a thin gold film on a glasssupport due to changes in the refractive index of the surface up to 300nm away. Biacore analysis generates association rate constants,dissociation rate constants, equilibrium dissociation constants, andaffinity constants. Binding affinity is obtained by assessing theassociation and dissociation rate constants using a Biacore surfaceplasmon resonance system (Biacore, Inc.). A biosensor chip is activatedfor covalent coupling of the target. The target is then diluted andinjected over the chip to obtain a signal in response units ofimmobilized material. Since the signal in resonance units (RU) isproportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix. Association anddissociation data are fit simultaneously in a global analysis to solvethe net rate expression for a 1:1 bimolecular interaction, yielding bestfit values for k_(on), k_(off) and R_(max) (maximal response atsaturation). Equilibrium dissociation constants for binding, K_(D)'s arecalculated from SPR measurements as k_(off)/k_(on).

In some embodiments, the anti-myostatin Adnectins of the inventionexhibit a K_(D) in the SPR affinity assay described in Example 6 of 500nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 150 nM orless, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nMor less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. Preferably,the K_(D) is 15 nM or less. More preferably, the K_(D) is 2.0 nM orless.

In some embodiments, the anti-myostatin Adnectins of the inventionexhibit an IC50 in the HTRF assay described in Example 4 of 5 nM orless, 4 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nMor less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, or 0.1 nM orless. Preferably, the IC50 is 1.5 nM or less. More preferably, the IC50is 0.5 nM or less.

In some embodiments, the anti-myostatin Adnectins of the inventionexhibit K_(D) in the kinetic exclusion assay described in Example 7 of 2nM or less, 1.5 nM or less, 1 nM or less, 900 pM or less, 850 pM orless, 800 pM or less, 750 pM or less, 700 pM or less, 650 pM or less,600 pM or less, 550 pM or less, 500 pM or less, 450 pM or less, 400 pMor less, 350 pM or less, 340 pM or less, 330 pM or less, 300 pM or less,250 pM or less, 200 pM or less, 150 pM or less, or 100 pM or less.Preferably, the K_(D) is 850 pM or less.

It should be understood that the assays described herein above areexemplary, and that any method known in the art for determining thebinding affinity between proteins (e.g., fluorescence based-transfer(FRET), enzyme-linked immunosorbant assay, and competitive bindingassays (e.g., radioimmunoassays)) can be used to assess the bindingaffinities of the anti-myostatin Adnectins of the invention.

In Vitro Assays for Antagonist Activity

The ability of anti-myostatin Adnectins to antagonize myostatin activitycan be readily determined using various in vitro assays. Preferably, theassays are high-throughput assays that allow for screening multiplecandidate Adnectins simultaneously. In some embodiments, the antagonisteffects of anti-myostatin Adnectins on myostatin activity can bedetermined in cell-based activin responsive element (ARE)-luciferasereporter assays, as described in Example 3. In certain embodiments, theanti-myostatin Adnectins of the invention decrease myostatin-inducedARE-luciferase activity by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or more relative to a control upon co-incubating myostatinwith an anti-myostatin Adnectin prior to stimulating cells with themixture. An exemplary control reaction involves treating cells withmyostatin alone or myostatin preincubated with an excess of a benchmarkmyostatin inhibitor such as Human Activin RIIB Fc Chimera (R&D Systems)or ActRIIb-Fc as described in Morrison et al. (Experimental Neurology2009; 217:258-68). In other embodiments, the anti-myostatin Adnectins ofthe invention inhibit ARE-luciferase reporter activity with an IC50 of500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nMor less, 50 nM or less, 10 nM or less, 5 nM or less, 1 nM, 0.5 nM orless, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.10 nM orless, as described in Example 3.

In other embodiments, the antagonistic effects of anti-myostatinAdnectins on myostatin activity can be determined by measuring theextent of SMAD phosphorylation in myostatin-treated cells, as describedin Example 5. In certain embodiments, the anti-myostatin Adnectins ofthe invention decrease myostatin-induced SMAD phosphorylation by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 97% or more relative to a control upon co-incubating myostatinwith an anti-myostatin Adnectin prior to stimulating the cells with themixture. An exemplary control reaction involves treating cells withmyostatin alone or myostatin preincubated with an excess of a benchmarkmyostatin inhibitor such as Human Activin RIIB Fc Chimera (R&D Systems)or ActRIIb-Fc as described in Morrison et al. (Experimental Neurology2009; 217:258-68). In some embodiments, the anti-myostatin Adnectins ofthe invention inhibit SMAD phosphorylation with an IC50 of 1 nM or less,0.8 nM or less, 0.6 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nMor less, or 0.1 nM or less in a 12-point or 4-point inhibition response,as described in Example 5. In other embodiments, the anti-myostatinAdnectins of the invention at 10 nM inhibit SMAD phosphorylation bymyostatin by at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, or at least 98% or more, asdescribed in Example 5.

Additionally, several in vitro model systems are known which use cells,tissue culture and histological methods for studying motoneuron disease.For example, a rat spinal cord organotypic slice subjected to glutamateexcitotoxicity is useful as a model system to test the effectiveness ofanti-myostatin Adnectins in preventing motor neuron degeneration. Corseet al., Neurobiol. Dis. (1999) 6:335 346. For a discussion of in vitrosystems for use in studying ALS, see, e.g., Bar, P. R., Eur. J.Pharmacol. (2000) 405:285 295; Silani et al., J. Neurol. (2000) 247Suppl 1:128 36; Martin et al., Int. J. Mol. Med. (2000) 5:3 13.

It should be understood that the assays described herein are exemplary,and that any method known in the art that can serve as a readout formyostatin activity are suitable for use for testing the myostatinantagonizing effects of the anti-myostatin Adnectins of the invention(e.g., real-time RT-PCR of mRNAs of SMAD target genes (e.g., Smad 7;Ciarmela et al., Journal of Clinical Endocrinology & Metabolism 2011;96; 755-65) or mRNAs of ARE-containing genes).

In Vivo Models

Various art-recognized animal models exist that recapitulate thesymptoms of diseases, disorders, and conditions associated with musclewasting associated, for example with muscular, neuromuscular,neurological and metabolic disorders. These models can be used to testthe efficacy of the anti-myostatin Adnectins of the invention.

For example, non-limiting examples of such animal models include, e.g.,the X-linked muscular dystrophy mouse (mdx) model (US2011/0008375,Gehrig et al., Nature 2012; 484:394-8), including 4 additional strainsof mdx mouse—mdx2cv, mdx3cv, mdx4cv, or mdx5cv mouse (Phelps et al.,Human Molecular Genetics. 1996; 5(8):1149-1153), the mdx mouse withadditional ablation of the dystrophin homologue utrophin (mdx/utr^(−/−))(Deconinck et al., Cell. 1997; 90(4):717-727), the alpha-SG-null C57BL/6mouse (Duclos et al. i(1998) J. Cell Biol. 142, 1461-1471), and thoserecently reviewed in Nakamura et al., (J Biomed Biotechnol. 2011;Article ID No: 184393), e.g., the mdx52 mouse, in which exon 52 of themurine DMD gene is deleted, the Golden Retriever muscular dystrophy(GRMD) model, the Canine X-Linked Muscular Dystrophy (CXMDj) model, andthe Hypertrophy Feline Muscular Dystrophy (HFMD) model (e.g., Shelton etal., Neuromuscular Disorders. 2005; 15(2):127-138).

Animal models for the study of motoneuron disorders such as ALS aretransgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase(SOD1) gene (mSOD1G93A and/or mSOD1G37R). These mice develop adominantly inherited adult-onset paralytic disorder with many of theclinical and pathological features of familial ALS. (e.g., Gurney etal., Science (1994) 264:1772 1775; Nagano et al., Life Sci (2002) 72:541548). Other animal models include two naturally occurring murine modelsfor progressive motor neuronopathy (pmn) and wobbler (Haegggeli andKato, Neurosci. Lett. (2002) 335:39 43). For a review of various animalmodels for use in studying motoneuron diseases such as ALS, see, e.g.,Jankowsky et al., Curr Neurol Neurosci. Rep. (2002) 2:457 464; Elliott,J. L., Neurobiol. Dis. (1999) 6:310 20; and Borchelt et al., BrainPathol. (1998) 8:735 757.

Animal models of other neurodegenerative or neuropathological diseasesin addition to ALS include a transgenic mouse model for evaluatingspinal and bulbar muscular atrophy (SBMA) (Katsuno et al., Neuron (2002)35:843 854), animal models for human paralytic poliomyelitis (Ford etal., Microb. Pathog. (2002) 33:97 107), animal models of spinal muscularatrophy (Schmid et al., J. Child Neurol. 22, 1004-1012, 2007), animalmodels for distal myopathy and hereditary inclusion body myopathy(Malicdan et al., Acta Myol. 2007 December; 26(3): 171-175), the murinemodel of genetic demyelinating disease (Suzuki et al., Microsc. Res.Tech. 1995; 32:204-214), and those described by Meyer ZuHörste et al.(Curr. Opin. Neurol. 2006; 19:464-473).

Animal models for testing the efficacy of the anti-myostatin Adnectinsof the invention against muscle volume loss due to atrophy and/orinactivity include, but are not limited to, mouse models of unilaterialimmobilization (Madaro et al., Basic Applied Myology 2008; 18:149-153),Achilles tendon laceration (tenotomy) (Bialek et al., Physiol Genomics2011; 43:1075-86), and those disclosed in Powers et al. (Am J PhysiolRegul Integr Comp Physiol 2005; 288:R337-44), such as, hindlimbsuspension of animals, limb immobilization, and controlled mechanicalventilation.

Relevant animal models for testing the efficacy of the anti-myostatinAdnectins of the invention in the treatment of metabolic disordersinclude, but are not limited to, those disclosed in Ramaro et al.(Indian J Med Res 2007; 125:451-472) and Kennedy et al. (Disease Models& Mechanisms 2010; 3:156-166), both of which are herein incorporated byreference in its entirety). Non-limiting examples of such animal modelsinclude Lep^(ob/ob) mice, Lepr^(db) mice, Kuo Kondo mice, KK/^(Ay) mice,New Zealand Obese (NZO) mice, NONcNZO10 mice, Tsumara Suzuki ObeseDiabetes (TSOD) and Tsumara Suzuki Non Obese (TSNO) mice, M16 mice,Zucker fatty rats, Zucker diabetic fatty rats, SHR/N-cp rat, JCR/LA-cprats, Otsuka Long Evans Tokushima Fatty rats, Obese rhesus monkeys,Cohen diabetic rats, Goto-Kakizaki rats, and non-obese mutant C57 BL/6(Akita) mice. Type 2 diabetes can also be induced by diet by, e.g.,feeding high fat feed to non-obese, non-diabetic C57BL6 mice (Surwit etal., Diabetes 1988; 37:1163-7). Type 2 diabetes can also be chemicallyinduced with, e.g., goldthioglucose (Le Marchand Brustel et al., Am JPhysiol 1978; 234:E348-58) or streptozotocin, or induced surgically(e.g., partial pancreatomized diabetic animals) (McNeil J H.,Experimental models of diabetes. Florida, US: CRC Press LLc; 1999;Sasaki et al., In Vivo 2000; 14:535-41). Many genetic animal models arealso known to recapitulate the symptoms and phenotypes of metabolicdisorders, such as those reviewed in Kennedy et al., 2010 (supra).

In some embodiments, the efficacy of the anti-myostatin Adnectins of theinvention for increasing muscle mass or volume can be tested bysubcutaneous injection of mice, as described in Example 9. Given thatthe inhibition of myostatin increases muscle mass, the anti-myostatinAdnectins of the invention are expected to increase body weight andmuscle mass, the extent to which can be used to determine the potency ofthe Adnectins.

In some embodiments, particularly when anti-myostatin Adnectins areimmunogenic in mice (e.g., due to the use of human fibronectin type IIIdomain) and chronic treatments are desired, the anti-myostatin Adnectinsof the invention can be administered to SCID mice, which are unable tomount cellular or humoral immune responses. In some embodiments, SCIDmice can be crossed with other genetic models, such as those describedherein (e.g., diabetic mice), to develop an immunocompromised mousemodel amenable to chronic treatment with the anti-myostatin Adnectins ofthe invention.

VIII. Therapeutic Applications

In one aspect, the present invention provides anti-myostatin Adnectinsuseful for the treatment of myostatin-related disease or disorders,e.g., muscle wasting disorders, muscle atrophy, metabolic disorders, andbone degenerative disorders. Accordingly, in certain embodiments theinvention provides methods for attenuating or inhibiting amyostatin-related disease or disorder in a subject comprisingadministering an effective amount of myostatin-binding polypeptide,i.e., an anti-myostatin Adnectin, to a subject. In some embodiments, thesubject is a human. In some embodiments, the anti-myostatin Adnectinsare pharmaceutically acceptable to a mammal, in particular a human. A“pharmaceutically acceptable” polypeptide refers to a polypeptide thatis administered to an animal without significant adverse medicalconsequences, such as essentially endotoxin free, or very low endotoxinlevels.

In some embodiments, the anti-myostatin Adnectins of the presentinvention will be administered to a subject in combination (concurrentlyor separately) with an agent known in the art to be useful for theparticular disorder or disease being treated.

In some embodiments, the target patient population for anti-myostatinAdnectin therapy is one that is not amenable to standard therapy for thedisease, disorder, or condition being treated due to, e.g., age,pre-existing conditions, genetic makeup, and/or co-morbidities. Theanti-myostatin Adnectins of the invention can serve as alternatives toexisting therapies that are associated with substantial side effects(e.g., reproductive performance) or safety concerns.

Exemplary diseases, disorders, and conditions for which theanti-myostatin Adnectins of the present invention will be useful aredescribed in more detail below.

Muscular, Neurological and Metabolic Diseases and Disorders

The anti-myostatin Adnectins of the present invention can be used totreat muscular, neurological and metabolic disorders associated withmuscle wasting and/or muscle atrophy. For example, myostatinoverexpression in vivo induces signs and symptoms characteristic ofcachexia, and myostatin binding agents can partially resolve the musclewasting effect of myostatin (Zimmers et al., Science 2002; 296:1486-8).Patients with AIDS also exhibit increased serum levels of myostatinimmunoreactive material compared to patients without AIDS or to AIDSpatients who do not exhibit weight loss (Gonzalez-Cadavid et al., PNAS1998; 95:14938-43). It has also been observed that heart-specificelimination of myostatin reduces skeletal muscle atrophy in mice withheart failure, and conversely, specifically overexpressing myostatin inthe heart is sufficient to induce muscle wasting (Breitbart et al.,AJP-Heart; 2011; 300:H1973-82). In contrast, myostatin knockout miceshow increased muscle mass, and an age-dependent decrease in fataccumulation compared to their wild type counterparts (McPherron et al.,J. Clin. Invest. 2002; 109:595-601).

Exemplary disorders that can be treated according to the methods of theinvention include myopathies and neuropathies, including, for example,motor neuron disease, neuromuscular and neurological disorders.

For example, anti-myostatin Adnectins can be used to treat inheritedmyopathies and neuromuscular disorders (e.g., muscular dystrophy(Gonzalez-Kadavid et al., PNAS, 1998; 95:14938-43), motor neurondisorders, congenital myopathies, inflammatory myopathies and metabolicmyopathies), as well as acquired myopathies (e.g., drug inducedmyopathy, toxin induced myopathy, infection induced myopathy,paraneoplastic myopathy and other myopathies associated with criticalillnesses).

Such disorders include, but are not limited to, Duchenne's musculardystrophy, progressive muscular dystrophy, Becker's type musculardystrophy, Dejerine-Landouzy muscular dystrophy, Erb's musculardystrophy, Emery Dreifuss muscular dystrophy, limb girdle musculardystrophy, oculopharyngeal muscular dystrophy (OPMD),facioscapulohumeral muscular dystrophy, congenital muscular dystrophy,infantile neuroaxonal muscular dystrophy, myotonic dystrophy (Steinert'sdisease), distal muscular dystrophy, nemaline myopathy, familialperiodic paralysis, nondystrophic myotonia, periodic paralyses, spinalmuscular atrophy, spinal muscular atrophy (SMA), amyotrophic lateralsclerosis (ALS), primary lateral sclerosis (PLS), progressive muscularatrophy (PMA), distal myopathy, myotubular/centronuclear myopathy,nemaline myopathy, mini core disease, central core disease,desminopathy, inclusion body myositis, dermatomyositis, polymyositis,mitochondrial myopathy, congenital myasthenic syndrome, myastheniagravis, post-polio muscle dysfunction, steroid myopathy, alcoholicmyopathy, perioperative muscular atrophy and ICU neuromyopathy.

Inherited and acquired neuropathies and radiculopathies which can betreated with anti-myostatin Adnectins include, but are not limited to,rigid spine syndrome, muscle-eye-brain disease, heredity motor andsensory neuropathy, Carcot-Marie-Tooth disease, chronic inflammatoryneuropathy, protressive hypertrophic neuropathy, tomaculous neuropathy,lupus, Guillain-Barre syndrome, chronic inflammatory demyelinatingpolyneuropathy, multiple sclerosis, sarcoidosis, diabetic neuropathy,alcoholic neuropathy, disease related neuropathies (e.g., HIV/AIDS, Lymedisease), toxin related neuropathies (e.g., heavy metal, chemotherapy),compression neuropathies (e.g., tumors, entrapment neuropathy), andneuropathies associated with injury or trauma (e.g., cauda equinesyndrome, paraplegia, quadriplegia).

In some embodiments, the anti-myostatin Adnectins of the invention canbe used to treat muscular dystrophies (e.g., Duchenne's musculardystrophy, Becker's type muscular dystrophy), ALS, and sarcopenia.

Additional disorders associated with muscle wasting that can be treatedwith the anti-myostatin Adnectins of the invention include cachexia,wasting syndrome, sarcopenia, congestive obstructive pulmonary disease,cystic fibrosis (pulmonary cachexia), cardiac disease or failure(cardiac cachexia), cancer, wasting due to AIDS, wasting due to renalfailure, renal disease, claudication, cachexia associated with dialysis,uremia, rheumatoid arthritis, muscle injury, surgery, repair of damagedmuscle, frailty, disuse atrophy, osteoporosis, osteoarthritis, ligamentgrowth and repair.

The methods of the invention can also be used to increase muscle volumein subjects who suffer from muscle atrophy due to disuse. Disuse atrophymay result from numerous causes including any disorder or state whichleads to prolonged immobility or disuse, including, but not limited toprolonged bedrest, being wheelchair bound, limb immobilization,unloading of the diaphragm via mechanical ventilation, solid organtransplant, joint replacement, stroke, CNS damage related weakness,spinal cord injury, recovery from severe burn, sedentary chronichemodialysis, post-trauma recovery, post-sepsis recovery and exposure tomicrogravity (Powers et al., Am J Physiol Regul Integr Comp Physiol2005; 288:R337-44).

In addition, age-related increases in fat to muscle ratios, andage-related muscular atrophy appear to be related to myostatin. Forexample, the average serum myostatin-immunoreactive protein increasedwith age in groups of young (19-35 yr old), middle-aged (36-75 yr old),and elderly (76-92 yr old) men and women, while the average muscle massand fat-free mass declined with age in these groups (Yarasheski et al. JNutr Aging 6(5):343-8 (2002)). Accordingly, Subjects with muscle atrophydue to aging, and/or subjects who are frail due to, for example,sarcopenia, would also benefit from treatment with the anti-myostatinAdnectins of the invention.

Also contemplated are methods for increasing muscle mass in food animalsby administering an effective dosage of the anti-myostatin Adnectins tothese animals. Since the mature C-terminal myostatin polypeptide isidentical in all species, anti-myostatin Adnectins would be expected toeffectively increase muscle mass and reducing fat in any agriculturallyimportant species, for example, but not limited to, cattle, chicken,turkeys, and pigs.

The efficacy of the anti-myostatin Adnectin in the treatment of musclewasting disorders or muscle atrophy can be determined, for example, byone or more methods for measuring an increase in muscle mass or volume,an increase in the number of muscle cells (hyperplasia), an increase inmuscle cell size (hypertrophy) and/or an increase in muscle strength.For example, the muscle volume increasing effects of the anti-myostatinAdnectins of the present invention are demonstrated in the Examplesdescribed infra. Methods for determining “increased muscle mass” arewell known in the art. For example, muscle content can be measuredbefore and after administration of an anti-myostatin Adnectin of theinvention using standard techniques, such as underwater weighing (see,e.g., Bhasin et al. New Eng. J. Med. (1996) 335:1-7) and dual-energyx-ray absorptiometry (see, e.g., Bhasin et al. Mol. Endocrinol. (1998)83:3155-3162). An increase in muscle size may be evidenced by weightgain of at least about 5-10%, preferably at least about 10-20% or more.

Metabolic Disorders

The anti-myostatin Adnectins of the present invention, which reducemyostatin activity and/or signaling, are useful for treating metabolicdisorders, such as obesity, type II diabetes mellitus, diabetesassociated disorders, metabolic syndrome, and hyperglycemia.

Myostatin is involved in the pathogenesis of type II diabetes mellitus.Myostatin is expressed in adipose tissue and myostatin deficient miceexhibit reduced fat accumulation as they age. Moreover, glucose load,fat accumulation, and total body weight are reduced in myostatin lackingagouti lethal yellow and obese (Lep^(ob/ob)) mice (Yen et al., FASEB J.8:479, 1994; McPherron et al., 2002). As disclosed in US2011/0008375,myostatin antagonists can decrease the fat to muscle ratio in an agedmouse model, preserve skeletal muscle mass and lean body mass, andattenuate kidney hypertrophy in STZ-induced diabetic mice.

As used herein, “obesity” is a condition in which excess body fat hasaccumulated to such an extent that health may be negatively affected. Itis commonly defined as a body mass index (BMI) of 30 kg/m2 or higherwhich distinguishes it from being overweight as defined by a BMI of 25kg/m2 or higher (see, e.g., World Health Organization (2000) (PDF).Technical report series 894: Obesity: Preventing and managing the globalepidemic. Geneva: World Health Organization). Excessive body weight isassociated with various diseases, particularly cardiovascular diseases,diabetes mellitus type II, obstructive sleep apnea, certain types ofcancer, and osteoarthritis.

A subject with obesity may be identified, for example, by determiningBMI (BMI is calculated by dividing the subject's mass by the square ofhis or her height), waist circumference and waist-hip ratio (theabsolute waist circumference (>102 cm in men and >88 cm in women) andthe waist-hip ratio (the circumference of the waist divided by that ofthe hips of >0.9 for men and >0.85 for women) (see, e.g., Yusuf S, etal., (2004). Lancet 364: 937-52), and/or body fat percentage (total bodyfat expressed as a percentage of total body weight: men with more than25% body fat and women with more than 33% body fat are obese; body fatpercentage can be estimated from a person's BMI by the followingformula: Bodyfat %=(1.2*BMI)+(0.23*age)−5.4−(10.8*gender), where genderis 0 if female and 1 if male). Body fat percentage measurementtechniques include, for example, computed tomography (CT scan), magneticresonance imaging (MRI), and dual energy X-ray absorptiometry (DEXA).

The term “type II diabetes” refers to a chronic, life-long disease thatresults when the body's insulin does not work effectively. A maincomponent of type II diabetes is “insulin resistance,” wherein theinsulin produced by the pancreas cannot connect with fat and musclecells to allow glucose inside to produce energy, causing hyperglycemia(high blood glucose). To compensate, the pancreas produces more insulin,and cells, sensing this flood of insulin, become even more resistant,resulting in a vicious cycle of high glucose levels and often highinsulin levels.

The phrase “disorders associated with diabetes” or “diabetes associateddisorders” or “diabetes related disorders,” as used herein, refers toconditions and other diseases which are commonly associated with orrelated to diabetes. Example of disorders associated with diabetesinclude, for example, hyperglycemia, hyperinsulinaemia, hyperlipidaemia,insulin resistance, impaired glucose metabolism, obesity, diabeticretinopathy, macular degeneration, cataracts, diabetic nephropathy,glomerulosclerosis, diabetic neuropathy, erectile dysfunction,premenstrual syndrome, vascular restenosis, ulcerative colitis, coronaryheart disease, hypertension, angina pectoris, myocardial infarction,stroke, skin and connective tissue disorders, foot ulcerations,metabolic acidosis, arthritis, and osteoporosis.

The efficacy of the anti-myostatin Adnectins in the treatment ofmetabolic disorders can be determined, for example, by one or moremethods of measuring an increase in insulin sensitivity, an increase inglucose uptake by cells from the subject, a decrease in blood glucoselevels, and a decrease in body fat.

For example, in subjects having type II diabetes or who are at risk ofdeveloping diabetes HbA1c levels can be monitored. The term “hemoglobin1AC” or “HbA1c” as used herein refers to the product of a non-enzymaticglycation of the hemoglobin B chain. The desired target range of HbA1clevels for people with diabetes can be determined from American DiabetesAssociation (ADA) guidelines, i.e., the Standards of Medical Care inDiabetes (Diabetes Care 2012; 35(Suppl 1):S511-563). Current HbA1ctarget levels are generally <7.0% for people with diabetes, and peoplewho do not have diabetes typically have HbA1c values of less than 6%.Accordingly, the efficacy of the anti-myostatin Adnectins of the presentinvention can be determined by an observed decrease in the HBA1c levelin a subject.

The methods of the invention further include administration of ananti-myostatin Adnectin alone, or in combination with other agents thatare known in the art for glycemic control (e.g., insulin, GLP1) or fortreating art-recognized diabetes-related complications.

Other Disorders

Myostatin knockout mice exhibit increased muscle mass, as well asincreased mineral content and density of the mouse humerus, andincreased mineral content of both trabecular and cortical bone atregions where muscles attach (Hamrick et al. Calcif Tissue Intl 2002;71:63-8). This suggests that increasing muscle mass may help improvebone strength and reduce osteoporosis and other degenerative bonediseases.

Additional diseases or disorders for which the anti-myostatin Adnectinsof the present invention are useful include wound healing, anti-fibroticdisease, Lambert-Eaton Syndrome, and Parkinson's Disease.

Combination Therapies

The anti-myostatin Adnectins provided herein may be employed incombination with anti-diabetic agents, anti-hyperglycemic agents,anti-hyperinsulinemic agents, anti-retinopathic agents, anti-neuropathicagents, anti-neurodegenerative agents, anti-nephropathic agents,anti-atherosclerotic agents, anti-ischemic agents, anti-hypertensiveagents, anti-obesity agents, anti-dyslipidemic agents, anti-dyslipidemicagents, anti-hyperlipidemic agents, anti-hypertriglyceridemic agents,anti-hypercholesterolemic agents, anti-restenotic agents,anti-pancreatic agents, lipid lowering agents, anorectic agents, memoryenhancing agents, anti-dementia agents, or cognition promoting agents,appetite suppressants, neuro- or musculo-restorative treatments,treatments for heart failure, treatments for peripheral arterial diseaseand anti-inflammatory agents.

The antidiabetic agents used in combination with the anti-myostatinAdnectins include, but are not limited to, insulin secretagogues orinsulin sensitizers, GPR40 receptor modulators, or other antidiabeticagents. These agents include, but are not limited to, dipeptidylpeptidase IV (DP4) inhibitors (for example, sitagliptin, saxagliptin,alogliptin, vildagliptin and the like), biguanides (for example,metformin, phenformin and the like), sulfonyl ureas (for example,gliburide, glimepiride, glipizide and the like), glucosidase inhibitors(for example, acarbose, miglitol, and the like), PPARγ agonists such asthiazolidinediones (for example, rosiglitazone, pioglitazone, and thelike), PPAR α/γ dual agonists (for example, muraglitazar, tesaglitazar,aleglitazar, and the like), glucokinase activators (as described in Fyfeet al., Drugs of the Future, 34(8):641-653 (2009) and incorporatedherein by reference), GPR119 receptor modulators (MBX-2952, PSN821,APD597 and the like), SGLT2 inhibitors (dapagliflozin, canagliflozin,remagliflozin and the like), amylin analogs such as pramlintide, and/orinsulin. Reviews of current and emerging therapies for the treatment ofdiabetes can be found in: Mohler et al., Medicinal Research Reviews,29(1):125-195 (2009), and Mizuno et al., Current Medicinal Chemistry,15:61-74 (2008).

The anti-myostatin Adnectins of the present invention may also beoptionally employed in combination with one or more hypophagic agentssuch as diethylpropion, phendimetrazine, phentermine, orlistat,sibutramine, lorcaserin, pramlintide, topiramate, MCHR1 receptorantagonists, oxyntomodulin, naltrexone, Amylin peptide, NPY Y5 receptormodulators, NPY Y2 receptor modulators, NPY Y4 receptor modulators,cetilistat, 5HT2c receptor modulators, and the like. The anti-myostatinAdnectins of the present invention may also be employed in combinationwith an agonist of the glucagon-like peptide-1 receptor (GLP-1 R), suchas exenatide, liraglutide, GPR-1(1-36) amide, GLP-1(7-36) amide,GLP-1(7-37) (as disclosed in U.S. Pat. No. 5,614,492 to Habener, thedisclosure of which is incorporated herein by reference), which may beadministered via injection, intranasally, or by transdermal or buccaldevices. Reviews of current and emerging therapies for the treatment ofobesity can be found in: Melnikova et al., Nature Reviews DrugDiscovery, 5:369-370 (2006); Jones, Nature Reviews: Drug Discovery,8:833-834 (2009); Obici, Endocrinology, 150(6):2512-2517 (2009); andElangbam, Vet. Pathol., 46(1):10-24 (2009).

The anti-myostatin Adnectins of the present invention can also beadministered with one or more additional therapeutic agents, asappropriate for the particular disease or disorder being treated.Non-limiting examples of additional agents include those that are usefulfor treating metabolic disorders such as type II diabetes and sarcopeniaand include, but are not limited to, GLP-1, GLP-1-like, amylin, andFGF21; those that are useful for treating anti-fibrotic disease,neuromuscular disease, motor neuron disease, and sarcopenia and include,but are not limited to, ghrelin, SARM, Riluzole, testosterone,androgens, growth hormone, hormone replacement therapy, COX-2inhibitors, troponin activators, J32 agonists, CTLA4-Ig (e.g.,abetacept, belatacept) and anti-TGFβ antibodies; those that are usefulfor treating cachexia and other wasting syndromes and include, but arenot limited to, TGFβ receptor kinase inhibitors, anti-IL-6, andubiquitin-proteasome inhibitors; those that are useful to treat musclecramps associated with myotonia and PLS include, but are not limited tophytoin, quinine, Baclofen and tizanidine; those that are useful forneuropathies include antidepressants (e.g., tricyclics and selectiveserotonin-norepinephrine re-uptake inhibitors (SNRI's)),anticonvulsants, cannbinoids, botulinum toxin Type A, NMDA antagonists(e.g., ketamine), dietary supplements (e.g., alpha lipoic acid andbenfotiamine); those that are useful for treating chronic inflammatoryneuropathies include, but are not limited to, corticosteroids,intravenous immunoglobulin, and immunosuppressive drugs (e.g.,cyclophosphamide, ciclosporin, azathiprine, mycophenolate mofetil,anti-thymocyte globulin, rituximab); and those that are useful fortreating Guillain Barré syndrome, sarcopenia, fracture, and bone lossand include, but are not limited to, Boniva (ibandronate) and PTH.

The anti-myostatin Adnectins of the present invention can beadministered with one or more additional agents used in symptomatictherapy. Non-limiting examples of such agents for treating the symptomsof ALS include mitochondrial permeability transition (MPT) poreactivators, fast skeletal troponin activators, macrophage regulators(e.g., NP001), lysosomal storage disease-treating agents (e.g., NP003),and nicotinic acetylcholine receptor (nAchR) antagonists. A non-limitingexample of an additional agent for use in treating the symptoms ofDMD/BMD is an agent that increases ATP levels.

The anti-myostatin Adnectins of the present invention can also beadministered with one or more additional agents used in diseasemodification therapy. Non-limiting examples of such agents for treatingALS include free radical scavengers (e.g., edaravone (norphenazone),CV-3611), VEGF agonists (e.g., sNN0029), Nogo-A protein I (e.g.,GSK122324), SOD1 inhibitors (e.g., ISIS-SOD1Rx), and PGE synthase 1inhibitors (e.g., AAD-2004). Non-limiting examples of such agents fortreating DMD/BMD include those that promote exon skipping (e.g.,antisense molecules such as drisapersen (PRO051/GSK2402968), PRO044,Eteplirsen, AVI-4658, AVI-5038, Ataluren (PTC124)), gene therapy agents,anti-inflammatory agents (e.g., CRD007), and anti-fibrotic agents (e.g.,HT-100).

As discussed above, agents that promote exon-skipping can be used incombination with the anti-myostatin Adnections of the present inventionfor treating Duchenne's muscular dystrophy and Becker's type musculardystrophy. Examples of specific exons that can be targeted to restorefunctional dystrophin include exons 7, 8, 17, 43, 44, 45, 46, 50, 51,52, 53, and 55 (see, e.g., Lu et al., Molecular Therapy 2011; 19:9-15).In some embodiments, more than one agent, e.g., antisenseoligonucleotides, can be used to induce multi-exon skipping.

IX. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositionscomprising an anti-myostatin Adnectin or fusion proteins thereofdescribed herein, wherein the composition is essentially endotoxin free,or at least contain no more than acceptable levels of endotoxins asdetermined by the appropriate regulatory agency (e.g., FDA).

Compositions of the present invention can be in the form of a pill,tablet, capsule, liquid, or sustained release tablet for oraladministration; a liquid for intravenous, subcutaneous or parenteraladministration; or a gel, lotion, ointment, cream, or a polymer or othersustained release vehicle for local administration.

Methods well known in the art for making compositions are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Compositions for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate compositions (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the composition varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC™ or polyethylene glycol (PEG).

The polypeptides of the present invention may be optionally administeredas a pharmaceutically acceptable salt, such as non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

The active ingredients may also be entrapped in a microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins of the invention, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated proteins of the invention may remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Compositions of the present invention for oral use include tabletscontaining the active ingredient(s) in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose and sorbitol),lubricating agents, glidants, and anti-adhesives (e.g., magnesiumstearate, zinc stearate, stearic acid, silicas, hydrogenated vegetableoils, or talc). Compositions for oral use may also be provided aschewable tablets, or as hard gelatin capsules wherein the activeingredient is mixed with an inert solid diluent, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

The compositions herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

X. Administration

A pharmaceutical composition comprising an anti-myostatin Adnectin ofthe present invention can be administered to a subject at risk for orexhibiting pathologies as described herein using standard administrationtechniques including oral, parenteral, pulmonary, transdermal,intramuscular, intranasal, buccal, sublingual, or suppositoryadministration. Preferably, administration of the anti-myostatinAdnectins the invention is parenteral. The term parenteral as usedherein includes intravenous, intramuscular, subcutaneous, rectal,vaginal, or intraperitoneal administration. Peripheral systemic deliveryby intravenous or intraperitoneal or subcutaneous injection ispreferred.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. An effective amount ofa pharmaceutical composition to be employed therapeutically will depend,for example, upon the therapeutic context and objectives. One skilled inthe art will appreciate that the appropriate dosage levels for treatmentwill thus vary depending, in part, upon the molecule delivered, theindication for which the binding agent molecule is being used, the routeof administration, and the size (body weight, body surface or organsize) and condition (the age and general health) of the patient.

For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The exact dosage will be determined in light of factors related to thesubject requiring treatment, and may be ascertained using standardtechniques. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy. In general, theanti-myostatin Adnectins of the present invention are administered atabout 0.01 mg/kg to about 50 mg/kg per day, preferably 0.01 mg/kg toabout 30 mg/kg per day, most preferably 0.01 mg/kg to about 20 mg/kg perday. In some embodiments, the anti-myostatin Adnectins of the presentinvention are administered at weekly dosages of about 1 to 50 mg, morepreferably about 10-50 mg. In other embodiments, the anti-myostatinAdnectins of the present invention are administered at monthly doses of30-200 mg, preferably 50-150 mg, and more preferably 60-120 mg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the binding agent molecule in the formulation used. Typically, acomposition is administered until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose or as multiple doses (at the same or differentconcentrations/dosages) over time, or as a continuous infusion. Furtherrefinement of the appropriate dosage is routinely made. Appropriatedosages may be ascertained through use of appropriate dose-responsedata. For example, the anti-myostatin Adnectin may be given daily (e.g.,once, twice, three times, or four times daily) or less frequently (e.g.,once every other day, once or twice weekly, or monthly). In addition, asis known in the art, adjustments for age as well as the body weight,general health, sex, diet, time of administration, drug interaction, andthe severity of the disease may be necessary, and will be ascertainablewith routine experimentation by those skilled in the art. Theanti-myostatin Adnectin is suitably administered to the patient at onetime or over a series of treatments.

Administration of an anti-myostatin Adnectin or a fusion thereof, andone or more additional therapeutic agents, whether co-administered oradministered sequentially, may occur as described above for therapeuticapplications. Suitable pharmaceutically acceptable carriers, diluents,and excipients for co-administration will be understood by the skilledartisan to depend on the identity of the particular therapeutic agentbeing administered.

XI. Methods of Detection and Diagnostics

The anti-myostatin Adnectins of the invention are also useful in avariety of diagnostic applications. For example, an anti-myostatinAdnectin of the invention may be used to diagnose a disorder or diseaseassociated with increased levels of myostatin. In a similar manner, ananti-myostatin Adnectin can be used in an assay to monitor myostatinlevels in a subject being treated for a myostatin-associated condition.The anti-myostatin Adnectins may be used with or without modification,and are labeled by covalent or non-covalent attachment of a detectablemoiety. The detectable moiety can be any one which is capable ofproducing, either directly or indirectly, a detectable signal. Forexample, the detectable moiety may be a radioisotope, such as H3, C14 or13, P32, S35, or 1131; a fluorescent or chemiluminescent compound, suchas fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme,such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods include conjugationchemistry well known in the art, including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a moiety (such as PEG) to a protein of theinvention, a linking group or reactive group is used. Suitable linkinggroups are well known in the art and include disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groupsand esterase labile groups. Preferred linking groups are disulfidegroups and thioether groups depending on the application. Forpolypeptides without a Cys amino acid, a Cys can be engineered in alocation to allow for activity of the protein to exist while creating alocation for conjugation.

Anti-myostatin Adnectins linked with a detectable moiety also are usefulfor in vivo imaging. The polypeptide may be linked to a radio-opaqueagent or radioisotope, administered to a subject, preferably into thebloodstream, and the presence and location of the labeled protein in thesubject is assayed. This imaging technique is useful in the staging andtreatment of malignancies. The protein may be labeled with any moietythat is detectable in a subject, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

Anti-myostatin Adnectins also are useful as affinity purificationagents. In this process, the polypeptides are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art.

Anti-myostatin Adnectins can be employed in any known assay method, suchas competitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting atarget molecule in a sample. A method may comprise contacting the samplewith an anti-myostatin Adnectins described herein, wherein saidcontacting is carried out under conditions that allow anti-myostatinAdnectin-target complex formation; and detecting said complex, therebydetecting said target in said sample. Detection may be carried out usingany art-recognized technique, such as, e.g., radiography, immunologicalassay, fluorescence detection, mass spectroscopy, or surface plasmonresonance. The sample may be from a human or other mammal. Theanti-myostatin Adnectins may be labeled with a labeling moiety, such asa radioactive moiety, a fluorescent moiety, a chromogenic moiety, achemiluminescent moiety, or a hapten moiety. The anti-myostatinAdnectins may be immobilized on a solid support.

XII. Kits and Articles of Manufacture

The anti-myostatin Adnectin of the invention can be provided in a kit, apackaged combination of reagents in predetermined amounts withinstructions for use in the therapeutic or diagnostic methods of theinvention.

For example, in one embodiment of the invention, an article ofmanufacture containing materials useful for the treatment or preventionof the disorders or conditions described above is provided. The articleof manufacture comprises a container and a label. Suitable containersinclude, for example, bottles, vials, syringes, and test tubes. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition of the invention which iseffective for preventing or treating the disorder or condition and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is ananti-myostatin Adnectin of the invention. The label on, or associatedwith, the container indicates that the composition is used for treatingthe condition of choice. The article of manufacture may further comprisea second container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Example 1—Protein Production

High Throughput Protein Production (HTPP)

Selected binders cloned into the PET9d vector upstream of a HIS₆tag andtransformed into E. coli BL21 DE3 plysS cells were inoculated in 5 ml LBmedium containing 50 μg/mL kanamycin in a 24-well format and grown at37° C. overnight. Fresh 5 ml LB medium (50 μg/mL kanamycin) cultureswere prepared for inducible expression by aspiration of 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-3-thiogalactoside (IPTG), the culture was expressed for 6hours at 30° C. and harvested by centrifugation for 10 minutes at 2750 gat 4° C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1-3 hours. Lysates were cleared and re-racked intoa 96-well format by transfer into a 96-well Whatman GF/D Unifilterfitted with a 96-well, 1.2 ml catch plate and filtered by positivepressure. The cleared lysates were transferred to a 96-well Nickel orCobalt-Chelating Plate that had been equilibrated with equilibrationbuffer (50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole, pH 8.0) and wereincubated for 5 min. Unbound material was removed by positive pressure.The resin was washed twice with 0.3 ml/well with Wash buffer #1 (50 mMNaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). Each wash wasremoved by positive pressure. Prior to elution, each well was washedwith 50 μl Elution buffer (PBS+20 mM EDTA), incubated for 5 min, andthis wash was discarded by positive pressure. Protein was eluted byapplying an additional 100 μl of Elution buffer to each well. After a 30minute incubation at room temperature, the plate(s) were centrifuged for5 minutes at 200 g and eluted protein collected in 96-well catch platescontaining 5 μl of 0.5 M MgCl₂ added to the bottom of elution catchplate prior to elution. Eluted protein was quantified using a totalprotein assay with wild-type ¹⁰Fn3 domain as the protein standard.

Expression and Purification of Insoluble Fibronectin-Based ScaffoldProtein Binders

For expression, selected clone(s), followed by the HIS₆tag, were clonedinto a pET9d vector and were expressed in E. coli BL21 DE3 plysS cells.Twenty ml of an inoculum culture (generated from a single plated colony)was used to inoculate 1 liter of LB medium or TB-Overnight ExpressionMedia (auto induction) containing 50 μg/ml Kanamycin and 34 μg/mlchloramphenicol. Cultures in LB medium were incubated at 37° C. untilA₆₀₀ 0.6-1.0 at which time they were induced with 1 mMisopropyl-β-thiogalactoside (IPTG) and grown for 4 hours at 30° C.Cultures grown in TB-Overnight Expression Media were incubated at 37° C.for 5 hours, at which time the temperature was lowered to 18° C. andgrown for 19 hours. Cultures were harvested by centrifugation for 30minutes at ≥10,000 g at 4° C. Cell pellets were frozen at −80° C. Afterthawing, the cell pellet was resuspended in 25 ml of lysis buffer (20 mMNaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease Inhibitor Cocktail-EDTA free(Roche), pH 7.4) using an Ultra-turrax homogenizer (IKA works) on ice.Cell lysis was achieved by high pressure homogenization (≥18,000 psi)using a Model M-110S Microfluidizer (Microfluidics). The insolublefraction was separated by centrifugation for 30 minutes at ≥23,300 g at4° C. The insoluble pellet recovered from centrifugation of the lysatewas washed with 20 mM sodium phosphate/500 mM NaCl, pH7.4. The pelletwas resolubilized in 6 M guanidine hydrochloride in 20 mM sodiumphosphate/500 mM NaCl pH 7.4 with sonication, followed by incubation at37 degrees for 1-2 hours. The resolubilized pellet was filtered with a0.45 μm filter and loaded onto a Histrap column equilibrated with the 20mM sodium phosphate/500 mM NaCl/6 M guanidine pH7.4 buffer. Afterloading, the column was washed for an additional 25 column volumes withthe same buffer. Bound protein was eluted with 50 mM imidazole in 20 mMsodium phosphate/500 mM NaCl/6 M guanidine-HCl, pH 7.4. The purifiedprotein was refolded by dialysis against 50 mM sodium acetate/150 mMNaCl, pH 4.5 or PBS, pH 7.2.

Expression and Purification of Soluble Fibronectin-Based ScaffoldProtein Binders

As an alternative to purification of insoluble binders, the purificationof soluble binders may also be used. For expression, selected clone(s),followed by the HIS₆tag, were cloned into a pET9d vector and expressedin E. coli BL21 DE3 plysS cells. Twenty ml of an inoculum culture(generated from a single plated colony) were used to inoculate 1 literof LB medium or TB-Overnight Expression Media (auto induction)containing 50 μg/ml Kanamycin and 34 μg/ml chloramphenicol. Cultures inLB medium were incubated at 37° C. until A₆₀₀ 0.6-1.0, followed byinduction with 1 mM isopropyl-β-thiogalactoside (IPTG) and grown for 4hours at 30° C. Cultures grown in TB-Overnight Expression Media wereincubated at 37° C. for 5 hours, after which the temperature was loweredto 18° C. and they were grown for 19 hours. Cultures were harvested bycentrifugation for 30 minutes at ≥10,000 g at 4° C. Cell pellets werefrozen at −80° C. The thawed cell pellet was resuspended in 25 ml oflysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), pH 7.4) using an Ultra-turrax homogenizer(IKA works) on ice. Cell lysis was achieved by high pressurehomogenization (≥18,000 psi) using a Model M-110S Microfluidizer(Microfluidics). The soluble fraction was separated by centrifugationfor 30 minutes at ≥23,300 g at 4° C. The supernatant was clarified usinga 0.45 m filter. The clarified lysate is loaded onto a Histrap column(GE) pre-equilibrated with 20 mM sodium phosphate/500 mM NaCl, pH 7.4.The column was then washed with 25 column volumes of the same buffer,followed by 20 column volumes of 20 mM sodium phosphate/500 mM NaCl/25mM imidazole, pH 7.4 and then 35 column volumes of 20 mM sodiumphosphate/500 mM NaCl/40 mM imidazole, pH 7.4. Protein was eluted with15 column volumes of 20 mM sodium phosphate/500 mM NaCl/500 mMimidazole, pH 7.4, fractions were pooled based on absorbance at A₂₈₀,and dialyzed against 1×PBS or 50 mM Tris, 150 mM NaCl, pH 8.5 or 50 mMNaOAc, 150 mM NaCl, pH4.5. Precipitates were removed by filtering with a0.22 μm filter.

Site-Specific PEGylation of Adnectins with Polyethylene Glycol (PEG)

Adnectins containing an engineered cysteine residue were conjugated withPEG or cysteine-blocking reagent via Michael-addition chemistry betweenthe thiol group on the cysteine and the maleimide functional group ofthe PEG or n-ethylmaleimide (NEM). For PEGylation with 2-branched 40 kDaPEG (NOF Corporation, P/N GL2-400MA), PEG was added in molar excess tothe protein solution under slightly acidic to neutral conditions. Thereaction was allowed to proceed at room temperature for 2 hours toovernight. The reaction was then applied to an ion exchange column toseparate the PEGylated Adnectin from the unreacted PEG-maleimide andnon-PEGylated Adnectin. For PEGylation with 4-branched 40 kDa PEG (NOF,P/N GL4-400MA) or 20 kDa bis-PEG (NOF corporation, P/N DE-200MA), theAdnectin was purified from SP FF in citrate buffer, pH 6.5. Followingreduction with DTT, the sample was desalted on a G25 column into thesame buffer to remove DTT and reacted with 20 kDa bis-PEG or 4-branched40K PEG at a 2:1 (PEG:adnectin) ratio for 2 hours at room temperature,and the reaction stopped with the addition of excess BME. The sample waspurified by a Resource 15S column (GE #17-0944-10) to selectively removeun-PEGylated species (and mon-PEGylated species in the case of 20 kDabis-PEG reaction). A final preparative SEC column (GE #17-1071-01,Superdex200, 26/60) was used (if needed) to remove high molecularspecies and unreactive Adnectin. To prepare a CYS-blocked Adnectin, a10-fold molar excess of NEM (Pierce Chemical) was added immediatelyafter the above mentioned G25 desalting step in citrate (pH 6.5) buffer.This was incubated for 1 hour at room temperature and the reactionstopped by the addition of excess BME. The sample was then extensivelydialysed against PBS. The purified conjugated adnectins were analyzed bySDS-PAGE and size exclusion chromatography.

Purification and PEGylation of Un-Tagged Fibronectin-Based ScaffoldProtein Binders

Selected binders were cloned into a pET9d vector with no HIS₆tag andexpressed in E. coli BL21 DE3 plysS cells. 25 ml of an inoculums culturepreviously isolated from a single plated colony were grown in a 125 mlflask until OD 600 nm reached 1-2, using pH 6.85 media+50 ug/mlkanamycin (Ammonium Chloride, Citric Acid, Ferric Ammonium Citrate,Magnesium Sulfate, Sodium Phosphate Monobasic Monohydrate, DextroseAnhydrous, Glycerol, Phytone Peptone, Yeast Extract Granulated,Kanamycin Sulfate, Ammonium Sulfate for pH adjustment). A 10 L fermentor(7.5 L starting volume of batch media) was inoculated at a final OD 600nm of 0.003. The culture was grown overnight at 25° C. with constantmixing at 650 rpm and dissolved 02 levels of >30%, while maintaining pH.The next day, the temperature was shifted to 37° C. and the culture wasgrown until OD 600 nm reached 20-25. Once the target OD was achieved,the temperature was shifted to 30° C. and the culture induced with IPTG(final concentration: 1 mM). A feed media (Glycerol, Phytone Peptone,Yeast Extract Granulated, Kanamycin Sulfate, and Phosphoric Acid for pHadjustment) was added at a rate of 40 ml media/L formation volume/hr.Cells were harvested by centrifugation at 10,000 g for 30 min at 4° C.Cell pellets were frozen at −80° C.

Cell paste was thawed in 1×PBS at a ratio of 10 ml buffer/g cell paste.Once thawed, the sample was disrupted with an UltraTurrax homogenizer(IKA works) until homogenous. The solution was then passed twice througha microfluidizer at 18,000 psi. The soluble fraction was separated bycentrifugation for 30 minutes at ≥10,000 g at 4° C. The supernatant wasdiluted 1:1 with sodium acetate (pH 4.5), and clarified with a 0.2 mfilter. The clarified lysate was loaded onto a SP FF column (SP1; GE)pre-equilibrated with 50 mM sodium acetate (pH 4.5). The column was thenwashed with 2 column volumes of the same buffer, followed by 8 columnvolumes of 50 mM sodium acetate/350 mM NaCl, pH 4.5. Protein was elutedwith 50 mM sodium acetate/700 mM NaCl, pH 4.5. Elutions were pooledbased on absorbance at A₂₈₀.

The SP1 elution was diluted 1:5 with 20 mM sodium phosphate (pH 6.7) andloaded onto a SP FF column (SP2) pre-equilibrated with 20 mM sodiumphosphate/100 mM NaCl, pH 6.7. The column was then washed with 2 columnvolumes of the same buffer. Protein was eluted from the column with 20mM sodium phosphate/0.5 M NaCl, pH 6.7. Elutions were pooled based onabsorbance at A₂₈₀.

The SP2 elution was diluted to 100 mM NaCl with 20 mM sodium phosphate(pH 6.7) and loaded onto a Q FF column (GE) pre-equilibrated with 20 mMsodium phosphate/100 mM NaCl, pH 6.7. The FT peak (containing product)was collected. The column was washed with equilibration buffer until theFT peak returned to baseline.

Adnectins containing an engineered cysteine residue were conjugated withPEG via Michael-addition chemistry between the thiol group on thecysteine and the maleimide functional group of the PEG reagent. The Q FTfraction was PEGylated with 40 kDa branched PEG at a molar ratio of 2:1PEG to protein. The sample was incubated overnight at room temperature.The PEGylation reaction was diluted with 2 parts 50 mM sodium acetate(pH 4.5) and loaded onto a SP FF column (GE) pre-equilibrated with 50 mMsodium acetate (pH 4.5). The column was washed with 2 column volumes ofthe same buffer. PEGylated protein was eluted from the column with 50 mMsodium acetate/200 mM NaCl, pH 4.5). Elutions were pooled based onabsorbance at A₂₈₀. PEGylated protein was concentrated using a 30 kDaMillipore Biomax membrane. The sample was filtered over a 0.22 μm filterand stored at, for example, 4° C., −20° C., or −80° C.

Transient Expression and Purification of Fc-Formatted Fibronectin-BasedScaffold Protein Binders

For DNA generation, selected candidates were cloned into a pDV-16plasmid from which E. coli Top10 cells were transformed. pDV-16 is amodified version of pTT5 (Yves Durocher, NRC Canada), wherein the humanIgG1-Fc coding sequence has been introduced, preceded by signalsequence, and restriction sites were included to allow insertion ofAdnectin coding sequences at either terminus of the Fc. Transformedcells were expanded by inoculating 1 L of Luria broth containing 100μg/ml Ampicillin and incubating in a rotating incubator at 225 rpm for18 hours at 37° C. Bacterial pellets were harvested by centrifugationat >10000 g for 30 minutes at 4° C. Purified plasmid DNA was isolatedusing a QIAGEN Plasmid Plus Mega Kit (QIAGEN) as described in themanufacturer's protocol. Purified DNA was quantified using absorbance at260 nm and frozen at −80° C. prior to use.

HEK 293-EBNA1 (clone 6E) (Yves Durocher, NRC Canada) cells were expandedto 2×10⁶ cells/ml in 2 L of F17 media in a 10 L GE Healthcare Wave bagat 37° C., 5% C02, and mixed by rocking at an 8 degree angle at 18 rpm.

DNA was prepared for transfection as follows: F17 media was warmed to37° C. DNA and a PEI transfection reagent were thawed in a sterilebiosafety hood. DNA (2.25 mg) was added to 100 ml of warmed F17 media ina sterile polypropylene culture flask and gently mixed by swirling. In aseparate flask, 6.75 mg of PEI (1 mg/ml) was combined with 100 ml ofpre-warmed F17 media and gently mixed by swirling. The flasks wereallowed to rest for 5 minutes prior to combining the contents by addingthe PEI solution to the flask containing the DNA and gently mixing byswirling.

The contents of the flask containing the DNA:PEI mixture were added tothe wave bag containing the HEK 293-6E cells after incubating at roomtemperature for 15 minutes in the biosafety hood. The bag containing thetransfected HEK 293-6E cells was incubated for twenty four hours at 37°C., 5% C02, and mixed by rocking at an 8 degree angle at 18 RPM. After24 hours, 100 ml of sterile filtered 20% Tryptone N1 (Organotechnie,Canada) dissolved in F17 media was aseptically added to the culture. Thecells and media were harvested after an additional 72 hours ofincubation as described above. Alternatively, transient HEK expressionin shake flasks (0.5 L media in a 2 L flask) can be performed with aDNA:PEI ratio of 1:2. Cells were separated from the conditioned media bycentrifugation at 6000 g for 30 minutes at 4° C. The conditioned mediawas retained, filtered through a 0.2 μM filter, and stored at 4° C.

The conditioned media was applied to a 10 ml chromatography columncontaining GE MabSelect Sure resin pre-equilibrated in PBS at a rate of5 ml/minute. After loading the filtered conditioned media, the columnwas washed with at least 100 ml of PBS at room temperature. The purifiedproduct was eluted from the column with the application of 100 mMGlycine/100 mM NaCl, pH 3.0. Fractions were neutralized in pH either bycollecting into tubes containing 1/6 volume of 1M Tris pH 8, or bypooling according to A280 absorbance followed by addition of 1M Tris pH8 to 100 mM. If the content of high molecular weight species is greaterthan 5% after Protein A elution, then the sample is further purified bya Superdex 200 (26/60) column (GE Healthcare) in PBS. The SEC fractionscontaining monomers are pooled and concentrated. The resulting protein Aor SEC pool was exhaustively dialyzed against PBS at 4° C., and sterilefiltered using a 0.22 μm cutoff filter prior to freezing at −80° C.

Bulk Manufacturing: Mammalian Expression and Primary Recovery: UCOE CHOSystem

A mammalian Research Cell Bank (RCB) was created by transfectinganti-myostatin Adnectin-Fc fusions cloned into the pUCOE vectorcontaining the Ubiquitous Chromatin Opening Element (UCOE) [ModifiedUCOE vector from Millipore] in CHO—S cells. An RCB was established byexpanding cells in selection media (0.04% (v/v) L-Glutamine (Invitrogen)and 0.01% (v/v) HT Supplement (Invitrogen) in CD CHO medium(Invitrogen)) containing 12.5 μg/mL puromycin. Low passage number cellswere aseptically isolated via centrifugation, resuspended in bankingmedia (0.04% (v/v) L-Glutamine (Invitrogen), 0.01% (v/v) HT Supplement(Invitrogen) and 7.5% (v/v) DMSO in CD CHO medium (Invitrogen)) to afinal concentration of 1×10⁷ cells/mL. These cells were initially frozenin a 70% isopropyl alcohol bath at −80° C. overnight and thentransferred to liquid nitrogen for long term storage the following day.

Cell culture was initiated by thawing a single RCB vial into 25 mL ofselection media containing 12.5 μg/mL puromycin and expanding theculture in the same media. Cells were allowed to reach a concentrationbetween 1-2×10⁶ cells/mL before being split back to 0.2×10⁶ cells/mL.Cells were generally maintained between 2-4 weeks prior to seeding abioreactor. The expansion culture was passaged a final time and allowedto grow to the point where a 15 L bioreactor containing 8 L ofproduction media (Invitrogen CD CHO media containing 0.01% (v/v) HTSupplement (Invitrogen), 0.04% (v/v) Glutamax (Gibco), and 0.005% (v/v)Pluronic F-68 (Gibco)) could be seeded at a final density of 0.2×10⁶cells/mL. The bioreactor culture was monitored daily for VCD (ViableCell Density), percent Viability, pH, and glucose concentration. Thebioreactor culture was fed on days 3 and 6 with a 10% total volume bolusaddition of Feed Media. The culture was harvested between Day 7 and Day9 with a percent viability>70%. During culture, the bioreactor culturewas controlled at a pH of 7.1, temperature of 37° C., % DO2 of 40%, anda constant RPM of 100.

On the day of harvest, the bioreactor cultures were directly passedthrough a 6.0/3.0 μm depth filter followed by a sterile 0.8/0.2 μmfiltration into a sterile bag. Clarified sterile culture was storedovernight at 2-8° C. The clarified culture was then concentrated viaflatsheet TFF using a 30,000 kDa membrane. The approximate concentrationwas 6×, depending on harvest titer. Concentrated supernatant was thensterile filtered into PETG bottles and either processed directly orstored at −80° C.

Anti-Myostatin-Adnectin-Fc Fusion Purification

Harvested culture supernatant (neat or concentrated) is loaded onto aMabSelect Protein A column previously equilibrated with PBS. Column iswashed with 5CV of 50 mM Tris pH8.0, 1M Urea, 10% PG. Adnectin-Fc fusionis eluted with 100 mM Glycine pH 3.3, collecting the peak into acontainer which is previously charged with 1CV of 200 mM Sodium AcetatepH 4.5. Peak elution is based on absorbance at A280.

The Protein A elution is diluted to pH 3.0 with the addition of 2 MCitric Acid and left at room temperature for 1 hour, for viralinactivation. Sample is then diluted with 200 mM Sodium PhosphateTribasic until pH 4.5 is reached. If necessary, the solution is furtherdiluted with water to lower conductivity below 10 ms/cm.

The diluted Protein A elution is passed over a Tosoh Q 600C AR (TosohBioscience), previously conditioned with 50 mM Sodium Acetate pH 4.5, ina negative capture mode. The flowthrough peak is collected, based onabsorbance at A280. The column is washed with 50 mM Sodium Acetate andstripped with 0.2N NaOH.

The Q 600C AR flowthrough is formulated using tangential flow filtrationutilizing a 30K NMWCO hollow fiber membrane (GE), with very gentlemixing of the retentate. The adnectin-Fc fusion is diafiltered into 25mM Sodium Phosphate 150 mM Trehaolse pH 7.0 for 6 diavolumes, and thenconcentrated to a target protein concentration.

Example 2—Biophysical Assessment of Anti-Myostatin Proteins

Size Exclusion Chromatography:

Standard size exclusion chromatography (SEC) was performed on candidateAdnectins resulting from the midscale process. SEC of midscaled materialwas performed using a Superdex 200 10/30 or on a Superdex 75 10/30column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system with UVdetection at A214 nm and A280 nm and with fluorescence detection(excitation 280 nm, emission 350 nm). A buffer of 100 mM sodiumsulfate/100 mM sodium phosphate/150 mM sodium chloride, pH 6.8 was usedat the appropriate flow rate for the SEC column employed. Gel filtrationstandards (Bio-Rad Laboratories, Hercules, Calif.) were used formolecular weight calibration. The results of the SEC on midscaledpurified Adnectins showed predominantly monomeric adnectin and elutionin the approximate range of 10 kDa vs. globular Gel Filtration standards(BioRad) as shown in Tables 9 and 10.

Thermostability:

Thermal Scanning Fluorescence (TSF) analysis of HTPP Adnectins wasperformed to screen them by relative thermal stability. Samples werenormalized to 0.2 mg/ml in PBS. 1 μl of Sypro orange dye diluted 1:40with PBS was added to 25 ul of each sample and the plate was sealed witha clear 96 well microplate adhesive seal. Samples were scanned using aBioRad RT-PCR machine by ramping the temperature from 25° C.−95° C., ata rate of 2 degrees per minute. The data was analyzed using BioRad CFXmanager 2.0 software. Th values obtained by TSF have been shown tocorrelate well with Tm values obtained by DSC over a melting range of40° C. to 70° C. This is considered the acceptable working range forthis technique. A result of ND (“No data”) is obtained when the slope ofthe transition curve is too small to allow its derivative peak (the rateof change in fluorescence with time) to be distinguished from noise. An“ND” result cannot be interpreted as an indication of thermostability.Differential Scanning Calorimetry (DSC) analyses of dialyzed HTPP'd andmidscaled Adnectins were performed to determine their respectiveT_(m)'s. A 0.5 mg/ml solution was scanned in a VP-Capillary DifferentialScanning calorimeter (GE Microcal) by ramping the temperature from 15°C. to 110° C., at a rate of 1 degree per minute under 70 p.s.i pressure.The data was analyzed vs. a control run of the appropriate buffer usinga best fit using Origin Software (OriginLab Corp). The results of theTSF and DSC analyses are summarized in Tables 8-10. As shown in Tables8-10, many of the clones exhibited unfolding temperatures of over 60°C., indicating a highly biophysically stable structure suitable formedicinal formulation. Adnectins were generally tolerant of PEGylationor Fc-formatting with no apparent loss in stability. In some cases,these formats afforded improved stability. For example, 3116_A07 as anunmodified Adnectin has a Tm by TSF of 60° C., but when PEGylated(ATI-1377) the Tm by DSC was 68° C., and in an Fc-X format (PRD-1286)the Tm by DSC was 66° C.

Example 3—Cell-Based Luciferase Assay

A luciferase reporter plasmid, Activin-Responsive Element (ARE)-luc, wasgenerated by ligating nine repeats of the ARE in tandem to the fireflyluciferase reporter. The plasmid was transiently transfected into HepG2cells. Plasmid pGL4.74[hRluc/TK] was co-transfected to normalize fortransfection efficiency. 10,000 cells were plated per well in a 96-wellplate. When a protein such as myostatin, activin, or BMP-11, is added tocells and binds to its cognate receptor, downstream SMAD signaling istriggered, leading to binding of a phosphorylated SMAD complex to theARE. The amount of, e.g., myostatin, exposed to the cells is directlyproportional to the amount of luciferase protein produced and,consequently, luciferase activity measured. When a myostatin antagonist(e.g., an anti-myostatin Adnectin) is added concurrently with myostatinto the cells, activation of the ARE decreases, leading to a decreasedluciferase production and activity.

In this experiment, (1) Anti-myostatin Adnectin and myostatin, (2)anti-myostatin Adnectin and activin A, or (3) anti-myostatin Adnectinand BMP-11 were preincubated prior to addition to cells. Myostatin (R&DSystems) was used at 10-500 pM, activin A (R&D Systems) at 10-500 pM,and BMP-11 (R&D Systems) at 10-500 pM. After overnight incubation withthese various combinations, cells were lysed and luciferase activity(luminescence) measured using the Dual-Glo Luciferase Assay System®(EnVision). The IC50 is defined as the concentration of Adnectinrequired to reach 50% inhibition of myostatin-induced ARE-luciferaseactivity.

As shown in Tables 8-10, anti-myostatin Adnectins inhibitedmyostatin-mediated increases in ARE-luc reporter activity.

Example 4—HTRF Binding Assay

An HTRF assay was used to measure the binding affinities ofanti-myostatin Adnectins to myostatin. The assay was a competitive HTRFassay using Eu-W1024 label as a donor fluorophore and Alexa Fluor® 647as an acceptor fluorophore. The biotinylated Adnectin 1889E01 and AlexaFluor® 647 labeled rhActRIIb-Fc can bind myostatin simultaneously at twodistinct binding sites. The Eu-W1024 labeled Streptavidin is used tobind biotinylated 1889E01. The two fluorophores, Eu-W1024 and AlexaFluor® 647, are brought together by the formation of a1889E01/myostatin/ActRIIb-Fc complex, and the HTRF signal can be read onan EnVision platereader (Perkin Elmer) using an HTRF protocol. In thepresence of a competitive Adnectin, the HTRF signal decreases. IC50s arepresented in Tables 8-10.

TABLE 8 Biophysical characterization, ARE-luciferase reporter assay, andHTRF binding assay results for anti-myostatin mono-Adnectins.ARE-luciferase assay HTRF Myo BMP-11 Activin A Myo ID Tm - TSF Tm-DSCIC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) 1979_B06 48 0.11 3.3 194 ND2062_G02 48 205 1500 >1500 ND 2522_C09 40 0.06 1.6 1000 ND 2523_G06 4949 4.2 46 >2000 ND 2524_C11 ND 55 0.1 0.89 765 ND 2524_D09 54 49 0.060.55 84 ND 2524_E10 ND 0.09 5.6 >1000 ND 2524_H05 40 0.09 7.3 >1000 ND2524_H11 49 0.03 6.2 >1000 ND 2525_B01 ND 0.11 3.2 73 ND 2525_D02 58 550.05 1.1 345 ND 2525_D05 ND 69 0.11 3.9 >1000 ND 2525_F07 46 0.133.4 >1000 ND 2987_A06 50 0.10 23 283 ND 2987_B04 48 0.12 3.3 239 ND2987_B09 52 0.01 0.92 172 ND 2987_C02 50 0.06 7.1 464 ND 2987_D05 490.11 2.4 2000 ND 2987_E03 51 0.10 2.3 224 ND 2987_E08 52 0.05 2.8 352 ND2987_F01 49 0.05 6.5 594 ND 2987_F06 52 0.08 20 538 ND 2987_G04 49 0.093.3 171 ND 2987_G09 54 0.05 0.91 >2000 ND 2987_H02 51 0.05 11 794 ND2987_H07 57 0.02 5.5 >400 ND 3006_A10 62 0.08 2.0 >2000 0.15 3007_B08 570.06 0.23 423 0.14 3007_C09 63 0.04 0.89 417 0.14 3007_C10 66 0.031.0 >2000 0.14 3008_A03 59 0.11 22.6 >2000 0.22 3008_B08 57 0.35 9.3 2540.37 3008_D04 56 0.09 1.2 720 0.14 3008_F01 63 0.08 0.12 >2000 0.213008_G01 57 0.03 0.31 >2000 0.11 3008_G03 58 0.09 1.3 >2000 0.133115_D04 64 0.16 3.6 >1000 0.20 3115_E06 62 0.07 2.0 >1000 0.14 3116_A0664 0.14 13 >1000 0.15 3116_A07 60 0.04 0.5 >1000 0.11 3116_C01 60 0.106.7 1000 0.35 3116_C06 61 0.14 8.9 >1000 0.18 3116_H06 60 0.10 1.6 >10000.13 3146_A08 69 0.70 48 >1000 0.26 ATI-1267 60 0.06 0.50 644 0.12ATI-1275 53 0.03 0.14 19 0.19 ATI-1277 no 0.14 1.18 2000 0.38 transitionATI-1340 54 0.05 4.87 324 0.16

TABLE 9 Biophysical characterization, ARE-luciferase reporter assay, andHTRF binding assay results for PEGylated anti-myostatin Adnectins. %ARE-luciferase assay HTRF Monomer Myo BMP-11 Activin A Myo ID Tm - TSFTm-DSC (SEC) IC50 (nM) IC50 (nm) IC50 (nM) IC50 (nM) ATI-1106 1771414 >2000 ATI-1107 63 98 19 888 >2000 ND ATI-1266 60 97.8 0.12 0.892000 0.21 ATI-1276 56 94.6 0.08 0.15 110 0.28 ATI-1278 46 93.7 0.271.1 >2000 0.54 ATI-1338 59 0.28 5.8 >1000 0.24 ATI-1339 61 0.286.0 >1000 0.28 ATI-1341 53 0.03 0.14 14 0.13 ATI-1359 57 96.5 0.263.1 >1000 0.36 ATI-1375 67 >99 0.17 1.6 >1000 0.76 ATI-1376 70 >99 0.030.83 >1000 0.32 ATI-1377 68 >99 0.05 0.78 >1000 0.15 ATI-1378 74 >990.29 5.4 >1000 1.29 ATI-1379 69 >99 0.10 4.3 >1000 0.28

TABLE 10 Biophysical characterization, ARE-luciferase reporter assay,and HTRF binding assay results for Fc-fused anti-myostatin Adnectins. %ARE-luciferase assay HTRF Monomer Myo BMP-11 Activin A Myo ID Tm - TSFTm-DSC (SEC) IC50 (nM) IC50 (nm) IC50 (nM) IC50 (nM) PRD-932 0.24 ND NDND PRD-1171 0.08 0.20 19 0.14 PRD-1173 0.02 0.10 6 0.12 PRD-1174 Tm1 610.04 0.10 4 0.09 Tm2 67 Tm3 83 PRD-1175 0.10 0.28 19 0.15 PRD-1177 0.160.28 21 0.64 PRD-1178 Tm1 60 0.08 0.25 16 0.27 Tm2 69 Tm3 84 PRD-1180Tm1 68 0.07 0.11 14 0.13 Tm2 83 PRD-1284 0.02 0.04 44 0.62 PRD-1285 Tm167 97 0.05 0.03 216 0.49 Tm2 68 Tm3 80 PRD-1286 Tm1 66 99 0.10 0.11 940.73 Tm2 68 Tm3 81 PRD-1287 0.30 0.80 >1000 2.70 PRD-1288 Tm1 65 93 0.080.10 >1000 0.47 Tm2 69 Tm3 81 PRD-1301 0.06 0.06 15 0.07 PRD-1302 0.030.02 14 0.12 PRD-1303 0.02 0.03 314 0.11 PRD-1304 0.05 0.07 45 0.24PRD-1305 0.06 0.07 113 0.10 PRD-1471 Tm1 62 99 0.16 0.16 60 0.47 Tm2 68Tm3 81 PRD-1472 Tm1 62 100 0.07 0.05 1000 0.41 Tm2 69 Tm3 80 PRD-1473Tm1 63 100 0.07 0.06 125 0.73 Tm2 69 Tm3 81 PRD-1474 Tm1 63 100 0.080.09 >1000 0.45 Tm2 69 Tm3 81

Example 5—Anti-Myostatin Adnectin-Mediated Inhibition ofMyostatin-Induced SMAD2 Phosphorylation

Human rhabdomyosarcoma RH41 cells (DSMZ, Braunschweig, Germany) wereused for the 12-, 2-, and 4-point inhibition response analysis describedbelow. Cells were removed from culture medium and washed to remove serumand quiesced in assay media containing BSA for 4 hours. Cells werelifted off the flask using versene and transferred to 96-well, V-bottompolypropylene plates at 5×10⁵ cells/well. For the 12-point inhibitionresponse, 100 pM recombinant myostatin (R&D Systems), preincubated for 1hour with a 5-fold dilution concentration range of Adnectins starting at1000 nM (i.e., 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM,0.0128 nM, 0.00256 nM, 0.000512 nM, 0.000102 nM, 0.0000204 nM), wasadded to the cells. For the 4-point inhibition response, 100 pM ofmyostatin, preincubated for 1 hour with a concentration range ofAdnectins (30 nM, 3 nM, 0.1 nM or 0.001 nM), was added to the cells. Forthe 2-point inhibition response, 100 pM of myostatin, preincubated for 1hour with a concentration range of Adnectins (10 nM or 0.5 nM), wasadded to the cells. Cells were treated with the myostatin-Adnectinmixture for 1 hour at 37° C. to induce SMAD2 phosphorylation (pSmad2).Stimulation was stopped by placing the cells on ice and adding ice-coldPBS. Cells were pelleted and lysed following standard protocols andSMAD2 phosphorylation detected using an ELISA assay (Cell SignalingTechnologies). The inhibition achieved by the concentration range ofAdnectins was plotted using GraphPad Prism Software and normalizing datapoints to controls which gave 100% and 0% inhibition. The IC50 isdefined as the concentration of Adnectin required to reach 50%inhibition of myostatin-induced SMAD2 phosphorylation. The datapresented in Table 11 indicate that Adnectins derived from affinityoptimization of parental clones 1979_B06 and 2062_G02 both potently andcompletely inhibited myostatin-induced pSMAD phosphorylation anddemonstrated IC50 values ranging from 0.78 nM to 0.06 nM. Thisrepresents a greater than 16-75 fold improvement in IC50 values over theparental clones 1979_B06 (IC50=12.8 nM) and 2062_G02 (IC50=59.1 nM).

TABLE 11 Inhibition of SMAD2 phosphorylation (pSMAD2) by anti-myostatinAdnectins pSmad2 Assay Myo IC50 (nM) Myo IC50 (nM) Myo % inhibition ID12-point 4-point at 10 nM 1979_B06 12.8 ± 2.4  (aka ATI-1133) 2062_G0259.1 ± 16.2 (aka ATI-1134) 2522_C09 0.13 2523_G06 0.78 2524_C11 0.142524_D09 0.11 2524_E10 0.13 2524_H05 0.13 2524_H11 0.11 2525_B01 0.142525_D02 0.36 2525_D05 0.20 2525_F07 0.27 3006_A10 51 3007_B08 973007_C09 80 3007_C10 76 3008_A03 87 3008_B08 90 3008_D04 92 3008_F01 863008_G01 98 3008_G03 91 3115_D04 0.06 3115_E06 0.06 3116_A06 0.273116_A07 0.06 3116_C01 0.26 3116_C06 0.73 3116_H06 0.06 3146_A08 0.78

Example 6—SPR Affinity Measurements for Anti-Myostatin Adnectins

Adnectin Binding Kinetics Using SPR Format A

Anti-human Fc antibody (Biacore/GE) was immobilized on a Biacore CM5chip via NHS/EDC coupling according to the manufacturer's instructions.ActRIIb-Fc (R&D Systems) was captured on both reference and active flowcells, followed by capture of human myostatin (R&D Systems), humanBMP-11 (GDF-11; R&D Systems), or human Activin A (R&D Systems) on activeflow cells only (each solubilized according to manufacturer's suggestedprotocol and diluted in HBSP running buffer). A concentration range ofanti-myostatin Adnectin was applied across all flow cells in HBSPrunning buffer. Regeneration of the chip surface between cycles wasaccomplished with two 30 second pulses of 3M MgCl₂. Kinetic traces ofreference-subtracted sensorgrams were fit to a 1:1 binding model usingBiaevaluation software. A summary of Biacore kinetic data is shown inTable 12.

The data shown in Table 12 indicate that optimized progeny Adnectinsbind myostatin tightly, with K_(D)s in the range of 0.06-1.47 nM,compared to parental Adnectins 1979_B06 and 2062_G02, which displayedK_(D)S of 29 and 49 nM, respectively.

Upon PEGylation, there is some loss in myostatin affinity, with K_(D)sranging from 0.76 to 14.4 nM, although there is no effect of PEGylationon potency in the ARE-luciferase assay (see Tables 8 and 9).

Adnectin selectivity over BMP-11 ranges from entirely non-selective toup to 17-fold, whereas binding to activin is either extremely weak ornon-existent, suggesting high selectivity over activin.

Adnectin Binding Kinetics Using SPR Format B (Useful for Fc-FormattedAdnectins)

Human myostatin (R&D Systems), human BMP-11 (GDF-11; R&D Systems), orhuman Activin A (R&D Systems) was solubilized according tomanufacturer-suggested protocol and immobilized on a Biacore CM5 chip at1-10 ug/mL in acetate (pH 4.0 or 4.5) buffer using standard NHS/EDCcoupling. A concentration range of anti-myostatin Adnectins was appliedin HBSP running buffer. Regeneration of the chip surface between cycleswas accomplished with 60 seconds of 10-50 mM NaOH. Kinetic traces ofreference-subtracted sensorgrams were fit to a 1:1 binding model usingBiaevaluation software. For Fc-formatted Adnectins, interaction kineticsare driven by avidity of bivalent Fc and dimeric myostatin even at lowimmobilization density. A summary of Biacore kinetic data is shown inTable 12. The data shown in Table 12 indicate that some of the Adnectinsrun in this SPR format bind myostatin and BMP-11 and also activin withsimilar affinities. The substantial selectivity over activin in theARE-luciferase assay, however, suggests the affinity for activin may beartificially accentuated in this SPR assay format.

TABLE 12 Summary of SPR kinetic data for anti-myostatin adnectins.Formats A and B are described in Example 6. Myo KD BMP-11 KD Activin AKD ID (nM) (nM) (nM) Format 1979_B06 29 489 no binding A, 25C (akaATI-1133) 2062_G02 48.8 697 no binding A, 25C (aka ATI-1134) 2522_C090.51 0.45 no/weak binding A, 25C 2523_G06 1.465 10.65 no binding A, 25C2524_C11 0.62 0.67 no binding A, 25C 2524_D09 0.64 0.81 weak binding A,25C 2524_E10 1.34 1.42 no binding A, 25C 2524_H05 0.88 0.87 weak bindingA, 25C 2524_H11 1.12 1.22 no binding A, 25C 2525_B01 1.29 1.58 weakbinding A, 25C 2525_D02 0.24 0.30 no/weak binding A, 25C 2525_D05 1.281.99 no binding A, 25C 2525_F07 0.79 0.97 no/weak binding A, 25C2987_H07 0.99 2.25 weak binding A, 25C ATI-1267 0.057 0.065 weak bindingA, 25C ATI-1275 0.15 0.15 weak binding A, 25C ATI-1277 0.16 0.14 weakbinding A, 25C ATI-1107 128 ~300 no binding A, 25C ATI-1266 0.76 2.57weak binding A, 25C ATI-1276 4.18 8.8 no binding A, 25C ATI-1278 6.043.3 no binding A, 25C ATI-1338 1.9 3.8 4.6 B, 37C ATI-1339 3.6 6.9 6.5B, 37C ATI-1359 7.9 35.45 weak binding A, 25C ATI-1375 12.6 33.2 weakbinding A, 25C ATI-1376 8.21 13.4 weak binding A, 25C ATI-1377 8.21 14.2weak binding A, 25C ATI-1378 14.4 59.6 weak binding A, 25C ATI-1379 9.521.6 weak binding A, 25C PRD-932 1.43 ND ND B, 25C PRD-1474 0.59 ND NDB, 37C (aka ATI-1465)

Example 7—Solution Phase Affinity for Anti-Myostatin Adnectins

The solution affinity of PRD-1474, an Fc-fused anti-myostatin Adnectin,for myostatin was measured using a Kinetic Exclusion Assay (KinExA).Quadruplicate titrations of PRD-1474 were performed with myostatin at amonomer concentration of 2 nM (n=2), 1 nM (n=1), and 0.7 nM (n=1). Therelative unbound myostatin concentration was measured by capture on anATI-1310 solid matrix (coupled to polyacrylamide beads via an engineeredfree cysteine) followed by detection with a fluorescent-labeledconstruct of the myostatin co-receptor, ActRIIB-Ig which can bindmyostatin simultaneously with the Adnectin. ATI-1310 is a relatedAdnectin which competes with PRD-1474 for binding to myostatin andallows for capture of unbound myostatin. The global Kd analysis shown inTable 13 gives a Kd of 170 pM with a 95% confidence interval of 330-60pM. The affinities of PRD-1177 and ATI-1338 were also measured using thesame assay format. Triplicate titrations of PRD-1177 were performed withmyostatin at a monomer concentration of 1 nM (n=2) and 0.8 nM (n=1).Triplicate titrations of ATI-1338 were performed with myostatin at amonomer concentration of 5 nM (n=1), 1.6 nM (n=1), and 1.4 nM (n=1).These analyses indicate that PRD-1177 binds myostatin with a global Kdvalue of 250 pM and a 95% confidence interval of 340-130 pM (Table 13).ATI-1338 binds myostatin with a global Kd value of 850 pM and a 95%confidence interval of 1400-330 pM.

TABLE 13 KinExA solution phase affinity measurements for bindingmyostatin. 95% confidence interval: Adnectin Kd Kd high Kd low PRD-1474170 pM 330 pM  60 pM PRD-1177 250 pM 340 pM 130 pM ATI-1338 850 pM 1400pM  330 pM

Example 8—Evaluation of Anti-Myostatin Adnectin Pharmacokinetics

To investigate the pharmacokinetic profile of Adnectins with differentPEGylated formats, anti-myostatin Adnectin 2987_H07 was formatted with2-branched 40 KD PEG (ATI-1338), 4-branched 40 KD PEG (ATI-1339), andBis 20 KD PEG (ATI-1341). Single dose studies of subcutaneousadministration with these three PEGylated Adnectins were conducted inC57BL6 mice. Total drug concentrations were determined by ELISA assay.Bioanalytical PK immunoassay for the quantitation of ATI-1338 used astandard sandwich format ELISA assay, where the 1338 was captured with amonoclonal antibody to HIS-TAG protein, then detected with a polyclonalanti-PEG antibody. As shown in Table 14, the two 40 KD PEGylatedformats, ATI-1338 and ATI-1339, provided more prominent pharmacokineticenhancement (i.e., longer half life (t_(1/2)) and higher dose-normalizedexposure) than the Bis-20 KD PEGylated format, ATI-1341.

TABLE 14 Pharmacokinectic Comparison of Three PEGylated Formats forAdnectin 2987_H07 PK Parameters ATI-1338 ATI-1339 ATI-1341 Dose (mg/kg)5 3.9 4 Cmax/Dose 547 370 113 (nM/(mg/kg)) Tmax (h) 24 24 4 AUC/Dose 3223 2.1 (nM*hr/(mg/kg)) t_(1/2) (h) 25 31 16

Single dose studies following intravenous and subcutaneousadministration of Fc-fused anti-myostatin Adnectins (PRD-1177, PRD-1286,and PRD-1474) were conducted in C57BL6 mice to evaluate the effects ofFc-fusion on pharmacokinetic parameters. Total drug concentrations weredetermined by ELISA assays. Bioanalytical PK immunoassay for thequantitation of the Fc conjugates for PRD1177, 1474, and 1286 all used astandard sandwich format ELISA assay using ECL technology, where the1177 was captured with a polyclonal antibody to the scaffold Adnectin,then detected with an anti-human IgG antibody. As shown in Table 15, allthree Fc-fused Adnectins had a longer half life (58-172 h) than thePEGylated Adnectin ATI-1338 (25 h). A lower SC bioavailability likelyreflects proteolysis during interstitial and lymphatic transit of thebiologic molecule. The SC bioavailability of the Fc-fused Adnectins arewithin a reasonable range based on published literature (e.g., Richteret al., AAPS J. 2012; 14:559-70).

TABLE 15 Pharmacokinectic Comparison of Three Fc-fused anti-myostatinAdnectins PK Parameters PRD-1177 PRD-1286 PRD-1474 Dose (mg/kg) 2 2 2Clearance 0.017 0.016 0.014 (mL/min/kg) Vdss (L/kg) 0.093 0.190 0.054t_(1/2) (h) 68 172 58 SC Bioavailability 60% 100% 94%

Example 9—Mechanism of Anti-Myostatin Adnectin Inhibition

Competitive binding assays to evaluate the ability of anti-myostatinAdnectins to compete with the ActRIIB receptor binding to myostatin werecarried out using competitive ELISA. Nunc Maxisorp plates were coatedwith 2 ug/mL ActRIIb-Fc (R&D Systems) in 0.2M sodium carbonate pH 9.6buffer overnight at 4° C. After washing with PBS-T (PBS containing 0.05%Tween-20), wells were blocked with OptEIA buffer (BD Biosciences) for 1h at 25° C. with shaking. Myostatin (10 nM; R&D Systems) waspreincubated with a concentration range of Adnectin or ActRIIb-Fccompetitor (0.2 pM to 1 uM) in OptEIA buffer for 1 h at 25° C. withshaking. The blocked and coated assay plate was washed with PBS-T, andthen myostatin/competitor mixtures were added and incubated for 30 minat 25° C. with shaking. The assay plate was washed with PBS-T, afterwhich bound myostatin was detected with 1:1000 biotinylated goatanti-myostatin polyclonal (R&D Systems) diluted in OptEIA, for 1 h at25° C. with shaking. After washing with PBS-T, 1:5000 Streptavidin-HRP(Thermo/Pierce) diluted in OptEIA was added, followed by incubation for30 min at 25° C. with shaking. The assay plate was developed with TMB(BD Biosciences), quenched with 2N sulfuric acid, and absorbance read atA450. As shown in FIG. 8, ActRIIb-Fc in solution fully blocks myostatinbinding to ActRIIb-Fc coated on the plate, as expected. In contrast,however, PRD-1288 (differs from PRD-1474 only in the linker sequence),PRD-1285, and PRD-1286 at concentrations up to 1 μM do not blockmyostatin from binding ActRIIb.

These data, taken together with the complete inhibition of myostatinsignaling for these same Adnectins in the ARE-luciferase assay atnanomolar concentrations (FIG. 9), demonstrate that the Adnectinmechanism of action at the cell surface must be downstream of myostatinbinding to ActRIIb, i.e., blockade of recruitment of the ALK4/5signaling receptor (FIG. 10). Since these Adnectins represent thesequence families exemplified in the current invention, and individualclones within a well-defined sequence family maintain the same bindingsite, the sequences covered by the current invention act by blockingALK4/5 recruitment to the myostatin-ActRIIb complex.

The pharmacokinetic data further indicate that myostatin-Adnectincomplex levels accumulate with time and that these complexes bind toActRIIb, thus acting as a dominant negative inhibitor of signalingindependent of free drug. This unique mechanism distinguishes theanti-myostatin Adnectins of the present invention from anti-myostatinantibodies described in the literature (e.g., U.S. Pat. No. 7,632,499),and indicate that the anti-myostatin Adnectins of the invention haveincreased activity.

Example 10—In Vivo Mouse Model of Musculoskeletal Efficacy

Male B6.SCID mice (9-13 weeks old, Jackson Laboratories, Bar Harbor,Me.) were housed in a temperature-controlled room with a reversed 12hour light/dark cycle. Water and standard chow food were available adlibitum. Mice were randomized and distributed between treatment groupsto receive either control or test compounds of the present inventionbased on body weight (about 20-22 g). In order to demonstrate in vivoefficacy of the compounds of the present invention, the compounds wereadministered either weekly (Fc-fusion anti-myostatin adnectins) or twicea week (PEGylated anti-myostatin adnectins) by subcutaneous injection.Test compounds were administered to the animals in Phosphate-BufferedSaline (PBS). Controls were treated with only reconstitution buffer.Test animals (n=8-10 mice/group) were dosed over a 14 day-time framesubcutaneously, with e.g., 5, 6 or 10 mg/kg/week of a compound of theinvention. Body weight measurements were recorded pre-randomization, onrandomization day, and two to three times a week during the treatmentperiods and at the end of the study. Lower leg muscle mass was recordedfrom body carcasses at the end of the study by quantitative magneticresonance imaging (MRI, Echo Medical Systems, Tex) analysis. Test groupswere compared to the control group. The results show that anti-myostatinAdnectins of the invention increased percent body weight from baseline(FIG. 11) and had significant anabolic effects on skeletal muscle volume(FIG. 12), compared to control mice (e.g., approximately a 7-10%increase in muscle volume compared to control.

Magnetic Resonance Imaging (MRI)

MRI for leg muscle volume measurements were performed on a BrukerPharmaScan 4.7 Tesla with a 16 cm bore (Bruker Biospin, Billerica, Ma.USA). A 62 mm volume coil was used for the transmitter and receiver.After collection of localizer images of the lower leg, T2 weightedimages were obtained using an axial slice plan. The fast spin-echo(RARE) sequence consisted of a 90° Hermite pulse followed by a 180°Hermite pulse with a TR/TE=2000/23 ms. Eleven axial slices werecollected from the top of the knee to the ankle with a matrix dimensionof 256×128 data points. The field of view was 5 cm by 2.5 cm, with a1.25 mm slice thickness and a RARE factor of 4 and 8 signal averages.Leg muscle volumes were calculated by summation of all axial slice areasmultiplied by the 1.25 mm slice thickness for total muscle volume ineach leg. Images were analyzed as an area average of theregion-of-interests (ROI) by Image Sequence Analysis (ISA, BrukerBiospin, Billerica, Ma.). Manual ROIs were drawn around the leg muscleexcluding the skin and subcutaneous fat area. The total average musclevolume for both legs is shown in FIG. 12.

MRI for heart volumes as a safety end point was also performed with thesame MRI scanner. After obtaining initial localizer images of thethoracic area, 9 axial images were collected from the great vessels tothe apex of the heart. Similar to the analysis of leg muscles, the axialareas were added and multiplied by the slice thickness of 1.25 mm toobtain the total heart volume for each animal. No significant change inheart volume was observed by MRI.

Statistics

Differences between groups were assessed using student's t-test 2 tailedpaired analysis.

Example 11—Efficacy of PRD-1474 on Muscle Growth In Vivo

Male B6.SCID mice (n=10/group) were maintained and treated as describedin Example 10, with the exception that PRD-1474 was administered at thevarious doses as indicated in FIG. 13, and the duration of treatment was28 days. PRD-1474 at 1 mg/kg showed a significant 11.1% increase inlower leg muscle volume compared to the PBS control group (p<0.0001).Significant increases in lower leg muscle volume of 27.7%, 29.7%, and32.8% were also observed with PRD-1474 at 10 mg/kg, 30 mg/kg, and 100mg/kg, respectively. No change in heart volume was observed in alltreatment dose groups relative to control. Data are presented asmean±standard deviation. The various dosage groups were compared usingANOVA. (*p<0.0001; #not significant between groups).

The data demonstrate that the anti-myostatin Adnectins of the inventionare effective at significantly lower dosages than myostatin inhibitorspreviously described (e.g., U.S. Pat. No. 7,632,499, J. Clin. Onclo.30(Suppl):Abstr. 2516, 2012). Thus, the anti-myostatin Adnectins of theinvention provide increased efficacy at lower dosages combined withdecreased undesired side effects, when administered alone or incombination with other myostatin inhibitors or other drugs, for treatingmuscle wasting and metabolic diseases described herein.

We claim:
 1. A pharmaceutical composition comprising a polypeptidecomprising a fibronectin type III tenth (¹⁰Fn3) domain which binds tomyostatin and comprises the amino acid sequence of SEQ ID NO: 6, wherein(Z)_(x), (Z)_(y), and (Z)_(z) comprise: (a) the amino acid sequences ofSEQ ID NOs: 7, 39, and 46, respectively; (b) the amino acid sequences ofSEQ ID NOs: 8, 40, and 47, respectively; (c) the amino acid sequences ofSEQ ID NOs: 12, 42, and 51, respectively; (d) the amino acid sequencesof SEQ ID NOs: 17, 39, and 56, respectively; (e) the amino acidsequences of SEQ ID NOs: 18, 39, and 57, respectively; (f) the aminoacid sequences of SEQ ID NOs: 17, 39, and 62, respectively; (g) theamino acid sequences of SEQ ID NOs: 32, 44, and 73, respectively; (h)the amino acid sequences of SEQ ID NOs: 33, 45, and 74, respectively;(i) the amino acid sequences of SEQ ID NOs: 34, 39, and 75,respectively; (j) the amino acid sequences of SEQ ID NOs: 35, 45, and74, respectively; or (k) the amino acid sequences of SEQ ID NOs: 38, 39,and 79, respectively, and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein (Z)_(x), (Z)_(y), and(Z)_(z) comprise the amino acid sequences of SEQ ID NOs: 34, 39, and 75.3. The pharmaceutical composition of claim 1, wherein the ¹⁰Fn3 domainfurther comprises an N-terminal extension sequence comprising the aminoacid sequence of SEQ ID NO:
 307. 4. The pharmaceutical composition ofclaim 1, wherein the ¹⁰Fn3 domain further comprises a C-terminalextension sequence comprising amino acid residues EI.
 5. Thepharmaceutical composition of claim 1, wherein the ¹⁰Fn3 domain furthercomprises an N-terminal extension sequence comprising the amino acidsequence of SEQ ID NO: 307, and a C-terminal extension sequencecomprising amino acid residues EI.
 6. The pharmaceutical composition ofclaim 5, wherein the ¹⁰Fn3 domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 275 and 277-282. 7.The pharmaceutical composition of claim 6, further comprising one ormore pharmacokinetic (PK) moieties selected from the group consisting ofpolyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serumalbumin, a serum albumin binding protein, and a serum immunoglobulinbinding protein.
 8. The pharmaceutical composition of claim 7, whereinthe PK moiety and the polypeptide are linked via a linker comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:181-187.
 9. The pharmaceutical composition of claim 7, wherein the PKmoiety is an Fc.
 10. The pharmaceutical composition of claim 9, whereinthe polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 252-273.
 11. The pharmaceutical composition ofclaim 10, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO: 273.